Compositions and methods for assessing eye vasculature

ABSTRACT

The present disclosure relates to compositions and methods for assessing blood vessels and organs of the body, more specifically to methods for assessing the vasculature of the eye.

This application claims the benefit of priority of U.S. ProvisionalApplication No. 62/160,338, filed May 12, 2015, the disclosure of whichis hereby incorporated by reference as if written herein in itsentirety.

The present disclosure relates to compositions and methods for assessingblood vessels and organs of the body, more specifically to methods forassessing the vasculature of the eye.

Angiography or arteriography is a medical imaging technique used tovisualize the inside, or lumen, of blood vessels and organs of the body,with particular interest in the arteries, veins, and the heart chambers.

Ocular angiography is a technique for examining the vasculature of theretina and choroid using a fluorescent dye and a specialized camera. Aneye angiogram helps eye doctors diagnose and manage the treatment ofretinal diseases. For example, the test may locate the presence ofabnormal blood vessels, blocked or leaking vessels, or identifyinflammation and tumors in the eye.

Photography of the circulatory system of the eye and angiography of theocular fundus requires introduction of a fluorescent dye into the bloodas it flows through the vasculature of the eye. Sodium fluorescein(NaF), carboxyfluorescein (CF), indocyanine green (ICG), lissaminegreen, patent blue, Evans blue, and acridine orange have been used inthe technique; however, sodium fluorescein is the only fluorescent dyecurrently in routine clinical use. Fluorescein and indocyanine greenangiography have been used in the study of bleeding vessels,neovascularity, tumors and ischemic tissues in a variety of disorders.

Sodium fluorescein has its light absorption peak near 490 nm and itfluoresces maximally at 514 nm to 520 nm. The molecular weight of sodiumfluorescein is 376 and it has a relatively high lipid solubility. Sodiumfluorescein is readily metabolized to fluorescein glucuronide, ametabolite that is significantly less fluorescent than the parent havingonly 5% of the fluorescent yield as fluorescein. Further, immediatelyupon intra-venous injection, fluorescein is largely converted to anon-fluorescent serum protein bound molecule (93%). Given these factors,angiograms taken with sodium fluorescein have a relatively short workinglife-time in the retinal vasculature. Also, with sodium fluorescein,vitreous leakage may obscure retinal and choroidal structures whichhinders photocoagulation therapy prior to or after angiography.Intravenous use of fluorescein may also cause adverse reactions such asnausea, vomiting, hives, acute hypotension, and anaphylaxis. One studyreported on the nature and frequency of moderate and severecomplications of intravenous fluorescein angiography. Adverse reactionswere scored as mild, moderate or severe, depending on the duration ofthe effect, the necessity of medical intervention, the time required forits resolution, and the final outcome. The frequency rate for moderatereaction was one out of sixty three (1:63), for severe reaction, one outof nineteen hundred (1:1900) and for death 1:222,000. In several otherstudies, more cases of severe anaphylactic shock as well as death havebeen reported.

Thus, there remains a need for improved compositions and methods forassessing blood vessels and organs of the body, more specifically tomethods for assessing the vasculature of the eye.

Accordingly, disclosed herein are improved compositions and methods forassessing blood vessels and organs of the body, more specifically tomethods for assessing the vasculature of the eye.

Provided is a method for visualizing the vasculature of a subject inneed thereof, comprising the steps of:

a. administering an effective amount of a compound of structural FormulaI

-   -    or a salt thereof; wherein        -   X¹ and X² are independently chosen from —CO(AA), —CN,            —CO₂R¹, —CONR²R³, —COR⁴, —NO₂, —SOR³⁵, —SO₂R⁶, —SO₂R⁷ and            —PO₃R⁸R⁹;        -   Y¹ and Y² are independently chosen from —OR¹⁰, —SR¹¹,            —NR¹²R¹³, —N(R¹⁴)COR¹⁵, —CONH(PS); —P(R¹⁶)₂, —P(OR¹⁷)₂ and

-   -    each Z¹ is independently chosen from a bond, —CR¹⁸R¹⁹—, —O—,        —NR²⁰—, —NCOR²¹—, —S—, —SO—, and —SO₂—;        -   each R¹ to R²¹ are independently chosen from hydrogen,            C₁-C₁₀ alkyl optionally substituted with hydroxyl and            carboxylic acid, C₃-C₆ polyhydroxylated alkyl, C₅-C₁₀ aryl,            C₅-C₁₀ heteroaryl, C₃-C₅ heterocycloalkyl optionally            substituted with C(O), —(CH₂)_(a)CO₂H optionally substituted            with C₅-C₁₀ heteroaryl, —(CH₂)_(a)CONR³⁰R³¹, —(CH₂)_(a)NHSO₃            ⁻, —(CH₂)_(a)NHSO₃H, —(CH₂)_(a)OH, —(CH₂)_(a)OPO₃ ⁼,            —(CH₂)_(a)OPO₃H₂, —(CH₂)_(a)OPO₃H⁻, —(CH₂)_(a)OR²²,            —(CH₂)_(a)OSO₃ ⁻, —(CH₂)_(a)OSO₃H, —(CH₂)_(a)PO₃ ⁼,            —(CH₂)_(a)PO₃H₂, —(CH₂)_(a)PO₃H⁻, —(CH₂)_(a)SO₃ ⁻,            —(CH₂)_(a)SO₃H, —(CH₂)_(d)CO(CH₂CH₂O)_(c)R²³,            —(CH₂)_(d)(CH₂CH₂O)_(c)R²⁴, —(CHCO₂H)_(a)CO₂H,            —CH₂(CHNH₂)_(a)CH₂NR²⁵R²⁶, —CH₂(CHOH)_(a)CO₂H,            —CH₂(CHOH)_(a)R²⁷, —CH[(CH₂)_(b)NH₂]_(a)CH₂OH,            —CH[(CH₂)_(b)NH₂]_(a)CO₂H, and —(CH₂)_(a)NR²⁸R²⁹;        -   each R²² to R³¹ are independently chosen from hydrogen,            C₁-C₁₀ alkyl, and C₁-C₅-dicarboxylic acid;        -   R³⁵ is chosen from C₁-C₁₀ alkyl optionally substituted with            hydroxyl and carboxylic acid, C₃-C₆ polyhydroxylated alkyl,            C₅-C₁₀ aryl, C₅-C₁₀ heteroaryl, C₃-C₅ heterocycloalkyl            optionally substituted with C(O), —(CH₂)_(a)CO₂H optionally            substituted with C₅-C₁₀ heteroaryl, —(CH₂)_(a)CONR³⁰R³¹,            —(CH₂)_(a)NHSO₃ ⁻, —(CH₂)_(a)NHSO₃H, —(CH₂)_(a)OH,            —(CH₂)_(a)OPO₃ ⁼, —(CH₂)_(a)OPO₃H₂, —(CH₂)_(a)OPO₃H⁻,            —(CH₂)_(a)OR²², —(CH₂)_(a)OSO₃ ⁻, —(CH₂)_(a)OSO₃H,            —(CH₂)_(a)PO₃ ⁼, —(CH₂)_(a)PO₃H₂, —(CH₂)_(a)PO₃H⁻,            —(CH₂)_(a)SO₃ ⁻, —(CH₂)_(a)SO₃H,            —(CH₂)_(d)CO(CH₂CH₂O)_(c)R²³, —(CH₂)_(d)(CH₂CH₂O)_(c)R²⁴,            —(CHCO₂H)_(a)CO₂H, —CH₂(CHNH₂)_(a)CH₂NR²⁵R²⁶,            —CH₂(CHOH)_(a)CO₂H, —CH₂(CHOH)_(a)R²⁷,            —CH[(CH₂)_(b)NH₂]_(a)CH₂OH, —CH[(CH₂)_(b)NH₂]_(a)CO₂H, and            —(CH₂)_(a)NR²⁸R²⁹;        -   (AA) is a polypeptide chain comprising one or more natural            or unnatural amino acids linked together by peptide bonds;        -   (PS) is a sulfated or non-sulfated polysaccharide chain            comprising one or more monosaccharide units connected by            glycosidic linkages; and        -   each ‘a’, ‘b’, and ‘d’ are independently chosen from 0 to            10, ‘c’ is chosen from 1 to 100 and each of ‘m’ and ‘n’            independently is an integer from 1 to 3;

b. irradiating the subject's vasculature with non-ionizing radiation,wherein the radiation causes the compound to fluoresce;

c. detecting the fluorescence of the compound in the subject'svasculature; and

d. visualizing the vasculature within the subject based on the detectedfluorescence.

Provided is a method of assessing the location of a disease or an injuryin a subject's vasculature, comprising the steps of:

a. administering an effective amount of a compound of structural FormulaI

-   -    or a salt thereof; wherein        -   X¹ and X² are independently chosen from —CO(AA), —CN,            —CO₂R¹, —CONR²R³, —COR⁴, —NO₂, —SOR³⁵, —SO₂R⁶, —SO₂OR⁷ and            —PO₃R⁸R⁹;        -   Y¹ and Y² are independently chosen from —OR¹⁰, —SR¹¹,            —NR¹²R¹³, —N(R¹⁴)COR¹⁵, —CONH(PS); —P(R¹⁶)₂, —P(OR¹⁷)₂ and

-   -    each Z¹ is independently chosen from a bond, —CR¹⁸R¹⁹—, —O—,        —NR²⁰—, —NCOR²¹—, —S—, —SO—, and —SO₂—;        -   each R¹ to R²¹ are independently chosen from hydrogen,            C₁-C₁₀ alkyl optionally substituted with hydroxyl and            carboxylic acid, C₃-C₆ polyhydroxylated alkyl, C₅-C₁₀ aryl,            C₅-C₁₀ heteroaryl, C₃-C₅ heterocycloalkyl optionally            substituted with C(O), —(CH₂)_(a)CO₂H optionally substituted            with C₅-C₁₀ heteroaryl, (CH₂)_(a)CONR³⁰R³¹, —(CH₂)_(a)NHSO₃            ⁻, —(CH₂)_(a)NHSO₃H, —(CH₂)_(a)OH, —(CH₂)_(a)OPO₃ ⁼,            —(CH₂)_(a)OPO₃H₂, —(CH₂)_(a)OPO₃H⁻, —(CH₂)_(a)OR²²,            —(CH₂)_(a)OSO₃ ⁻, —(CH₂)_(a)OSO₃H, —(CH₂)_(a)PO₃ ⁼,            —(CH₂)_(a)PO₃H₂, —(CH₂)_(a)PO₃H⁻, —(CH₂)_(a)SO₃ ⁻,            —(CH₂)_(a)SO₃H, —(CH₂)_(d)CO(CH₂CH₂O)_(c)R²³,            —(CH₂)_(d)(CH₂CH₂O)_(c)R²⁴, —(CHCO₂H)_(a)CO₂H,            —CH₂(CHNH₂)_(a)CH₂NR²⁵R²⁶, —CH₂(CHOH)_(a)CO₂H,            —CH₂(CHOH)_(a)R²⁷, —CH[(CH₂)_(b)NH₂]_(a)CH₂OH,            —CH[(CH₂)_(b)NH₂]_(a)CO₂H, and —(CH₂)_(a)NR²⁸R²⁹;        -   each R²² to R³¹ are independently chosen from hydrogen,            C₁-C₁₀ alkyl, and C₁-C₅-dicarboxylic acid;        -   R³⁵ is chosen from C₁-C₁₀ alkyl optionally substituted with            hydroxyl and carboxylic acid, C₃-C₆ polyhydroxylated alkyl,            C₅-C₁₀ aryl, C₅-C₁₀ heteroaryl, C₃-C₅ heterocycloalkyl            optionally substituted with C(O), —(CH₂)_(a)CO₂H optionally            substituted with ₅C₁₀ heteroaryl, —(CH₂)_(a)CONR³⁰R³¹,            —(CH₂)_(a)NHSO₃ ⁻, —(CH₂)_(a)NHSO₃H, —(CH₂)_(a)OH,            —(CH₂)_(a)OPO₃ ⁼, —(CH₂)_(a)OPO₃H₂, —(CH₂)_(a)OPO₃H⁻,            —(CH₂)_(a)OR²², —(CH₂)_(a)OSO₃ ⁻, —(CH₂)_(a)OSO₃H,            —(CH₂)_(a)PO₃ ⁼, —(CH₂)_(a)PO₃H₂, —(CH₂)_(a)PO₃H⁻,            —(CH₂)_(a)SO₃ ⁻, —(CH₂)_(a)SO₃H,            —(CH₂)_(d)CO(CH₂CH₂O)_(c)R²³, —(CH₂)_(d)(CH₂CH₂O)_(c)R²⁴,            —(CHCO₂H)_(a)CO₂H, —CH₂(CHNH₂)_(a)CH₂NR²⁵R²⁶,            —CH₂(CHOH)_(a)CO₂H, —CH₂(CHOH)_(a)R²⁷,            —CH[(CH₂)_(b)NH₂]_(a)CH₂OH, —CH[(CH₂)_(b)NH₂]_(a)CO₂H, and            —(CH₂)_(a)NR²⁸R²⁹;        -   (AA) is a polypeptide chain comprising one or more natural            or unnatural amino acids linked together by peptide bonds;        -   (PS) is a sulfated or non-sulfated polysaccharide chain            comprising one or more monosaccharide units connected by            glycosidic linkages; and        -   each ‘a’, ‘b’, and ‘d’ are independently chosen from 0 to            10, ‘c’ is chosen from 1 to 100 and each of ‘m’ and ‘n’            independently is an integer from 1 to 3;

b. irradiating the subject's vasculature with non-ionizing radiation,wherein the radiation causes the compound to fluoresce;

c. detecting the fluorescence of the compound in the subject'svasculature; and

d. assessing the location of disease or injury in the subject'svasculature, based on the detected fluorescence.

Provided is kit for assessing the vasculature in a subject in needthereof, comprising:

a. a compound of structural Formula I

-   -    or a salt thereof; wherein        -   X¹ and X² are independently chosen from —CO(AA), —CN,            —CO₂R¹, CONR²R³, —COR⁴, —NO₂, —SOR³⁵, —SO₂R⁶, —SO₂OR⁷ and            —PO₃R⁸R⁹;        -   Y¹ and Y² are independently chosen from —OR¹⁰, —SR¹¹,            —NR¹²R¹³, —N(R¹⁴)COR¹⁵, —CONH(PS); —P(R¹⁶)₂, —P(OR¹⁷)₂ and

-   -    each Z¹ is independently chosen from a bond, —CR¹⁸R¹⁹—, —O—,        —NR²⁰—, —NCOR²¹—, —S—, —SO—, and —SO₂—;        -   each R¹ to R²¹ are independently chosen from hydrogen,            C₁-C₁₀ alkyl optionally substituted with hydroxyl and            carboxylic acid, C₃-C₆ polyhydroxylated alkyl, C₅-C₁₀ aryl,            C₅-C₁₀ heteroaryl, C₃-C₅ heterocycloalkyl optionally            substituted with C(O), —(CH₂)_(a)CO₂H optionally substituted            with C₅-C₁₀ heteroaryl, —(CH₂)_(a)CONR³⁰R³¹, —(CH₂)_(a)NHSO₃            ⁻, —(CH₂)_(a)NHSO₃H, —(CH₂)_(a)OH, —(CH₂)_(a)OPO₃ ⁼,            —(CH₂)_(a)OPOH₂, —(CH₂)_(a)OPO₃H⁻, —(CH₂)_(a)OR²²,            —(CH₂)_(a)OSO₃ ⁻, —(CH₂)_(a)OSO₃H, —(CH₂)_(a)PO₃ ⁼,            —(CH₂)_(a)PO₃H₂, —(CH₂)_(a)PO₃H⁻, —(CH₂)_(a)SO₃ ⁻,            —(CH₂)_(a)SO₃H, —(CH₂)_(d)CO(CH₂CH₂O)_(c)R²³,            —(CH₂)_(d)(CH₂CH₂O)_(c)R²⁴, —(CHCO₂H)_(a)CO₂H,            —CH₂(CHNH₂)_(a)CH₂NR²⁵R²⁶, —CH₂(CHOH)_(a)CO₂H,            CH₂(CHOH)_(a)R²⁷, —CH[(CH₂)_(b)NH₂]_(a)CH₂OH,            —CH[(CH₂)_(b)NH₂]_(a)CO₂H, and —(CH₂)_(a)NR²⁸R²⁹;        -   each R²² to R³¹ are independently chosen from hydrogen,            C₁-C₁₀ alkyl, and C₁-C₅-dicarboxylic acid;        -   R³⁵ is chosen from C₁-C₁₀ alkyl optionally substituted with            hydroxyl and carboxylic acid, C₃-C₆ polyhydroxylated alkyl,            C₅-C₁₀ aryl, C₅-C₁₀ heteroaryl, C₃-C₅ heterocycloalkyl            optionally substituted with C(O), —(CH₂)_(a)CO₂H optionally            substituted with C₅-C₁₀ heteroaryl, —(CH₂)_(a)CONR³⁰R³¹,            —(CH₂)_(a)NHSO₃ ⁻, —(CH₂)_(a)NHSO₃H, —(CH₂)_(a)OH,            —(CH₂)_(a)OPO₃ ⁼, —(CH₂)_(a)OPO₃H₂, —(CH₂)_(a)OPO₃H⁻,            —(CH₂)_(a)OR²², —(CH₂)_(a)OSO₃ ⁻, —(CH₂)_(a)OSO₃H,            —(CH₂)_(a)PO₃ ⁼, —(CH₂)_(a)PO₃H₂, —(CH₂)_(a)PO₃H⁻,            —(CH₂)_(a)SO₃ ⁻, —(CH₂)_(a)SO₃H,            —(CH₂)_(d)CO(CH₂CH₂O)_(c)R²³, —(CH₂)_(d)(CH₂CH₂O)_(c)R²⁴,            —(CHCO₂H)_(a)CO₂H, —CH₂(CHNH₂)_(a)CH₂NR²⁵R²⁶,            —CH₂(CHOH)_(a)CO₂H, —CH₂(CHOH)_(a)R²⁷,            —CH[(CH₂)_(b)NH₂]_(a)CH₂OH, —CH[(CH₂)_(b)NH₂]_(a)CO₂H, and            —(CH₂)_(a)NR²⁸R²⁹;        -   (AA) is a polypeptide chain comprising one or more natural            or unnatural amino acids linked together by peptide bonds;        -   (PS) is a sulfated or non-sulfated polysaccharide chain            comprising one or more monosaccharide units connected by            glycosidic linkages; and        -   each ‘a’, ‘b’, and ‘d’ are independently chosen from 0 to            10, ‘c’ is chosen from 1 to 100 and each of ‘m’ and ‘n’            independently is an integer from 1 to 3; and

b. written instructions for assessing the vasculature in the subject,comprising the steps of:

-   -   i. administering an effective amount of the compound of        structural Formula I;    -   ii. irradiating the subject's vasculature with non-ionizing        radiation, wherein the radiation causes the compound to        fluoresce;    -   iii. detecting the fluorescence of the compound in the subject's        vasculature; and

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. shows retinal imaging at 2 minutes following IV administrationof fluorescein (left) or Example 2 (right) in mice.

DETAILED DESCRIPTION Abbreviations And Definitions

To facilitate understanding of the disclosure, a number of terms andabbreviations as used herein are defined below as follows:

When introducing elements of the present disclosure or the embodiment(s)thereof, the articles “a”, “an”, “the” and “said” are intended to meanthat there are one or more of the elements. The terms “comprising”,“including” and “having” arc intended to be inclusive and mean thatthere may be additional elements other than the listed elements.

The term “and/or” when used in a list of two or more items, means thatany one of the listed items can be employed by itself or in combinationwith any one or more of the listed items. For example, the expression “Aand/or B” is intended to mean either or both of A and B, i.e. A alone, Balone or A and B in combination. The expression “A, B and/or C” isintended to mean A alone, B alone, C alone, A and B in combination, Aand C in combination, B and C in combination or A, B, and C incombination.

When ranges of values are disclosed, and the notation “from n₁ . . . ton₂” or “between n₁ . . . and n₂” is used, where n₁ and n2 are thenumbers, then unless otherwise specified, this notation is intended toinclude the numbers themselves and the range between them. This rangemay be integral or continuous between and including the end values. Byway of example, the range “from 2 to 6 carbons” is intended to includetwo, three, four, five, and six carbons, since carbons come in integerunits. Compare, by way of example, the range “from 1 to 3 μM(micromolar),” which is intended to include 1 μM, 3 μM, and everythingin between to any number of significant figures (e.g., 1.255 μM, 2.9999μM, etc.).

The term “about,” as used herein, is intended to qualify the numericalvalues that it modifies, denoting such a value as variable within amargin of error. When no particular margin of error, such as a standarddeviation to a mean value given in a chart or table of data, is recited,the term “about” should be understood to mean that range which wouldencompass the recited value and the range which would be included byrounding up or down to that figure as well, taking into accountsignificant figures.

The term “acyl,” as used herein, alone or in combination, refers to acarbonyl attached to an alkenyl, alkyl, aryl, cycloalkyl, heteroaryl,heterocycle, or any other moiety were the atom attached to the carbonylis carbon. An “acetyl” group refers to a —C(O)CH₃ group. An“alkylcarbonyl” or “alkanoyl” group refers to an alkyl group attached tothe parent molecular moiety through a carbonyl group. Examples of suchgroups include methylcarbonyl and ethylcarbonyl. Examples of acyl groupsinclude formyl, alkanoyl and aroyl.

The term “alkenyl,” as used herein, alone or in combination, refers to astraight-chain or branched-chain hydrocarbon radical having one or moredouble bonds and containing from 2 to 20 carbon atoms. In certainembodiments, the alkenyl will comprise from 2 to 6 carbon atoms. Theterm “alkenylene” refers to a carbon-carbon double bond system attachedat two or more positions such as ethenylene [(—CH═CH—),(—C::C—)].Examples of suitable alkenyl radicals include ethenyl, propenyl,2-methylpropenyl, 1,4-butadienyl and the like. Unless otherwisespecified, the term “alkenyl” may include “alkenylene” groups.

The term “alkoxy,” as used herein, alone or in combination, refers to analkyl ether radical, wherein the term alkyl is as defined below.Examples of suitable alkyl ether radicals include methoxy, ethoxy,n-propoxy, isopropoxy, n-butoxy, iso-butoxy, sec-butoxy, tert-butoxy,and the like.

The term “alkyl,” as used herein, alone or in combination, refers to astraight-chain or branched-chain alkyl radical containing from 1 to 20carbon atoms. In certain embodiments, the alkyl will comprise from 1 to10 carbon atoms. In further embodiments, the alkyl will comprise from 1to 6 carbon atoms. Alkyl groups may be optionally substituted as definedherein. Examples of alkyl radicals include methyl, ethyl, n-propyl,isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, pentyl, iso-amyl,hexyl, octyl, nonyl and the like. The term “alkylene,” as used herein,alone or in combination, refers to a saturated aliphatic group derivedfrom a straight or branched chain saturated hydrocarbon attached at twoor more positions, such as methylene (—CH2-). Unless otherwisespecified, the term “alkyl” may include “alkylene” groups.

The term “alkylamino,” as used herein, alone or in combination, refersto an alkyl group attached to the parent molecular moiety through anamino group. Suitable alkylamino groups may be mono-or dialkylated,forming groups such as, for example, N-methylamino, N-ethylamino,N,N-dimethylamino, N,N-ethylmethylamino and the like.

The term “alkylidene,” as used herein, alone or in combination, refersto an alkenyl group in which one carbon atom of the carbon-carbon doublebond belongs to the moiety to which the alkenyl group is attached.

The term “alkylthio,” as used herein, alone or in combination, refers toan alkyl thioether (R—S—) radical wherein the term alkyl is as definedabove and wherein the sulfur may be singly or doubly oxidized. Examplesof suitable alkyl thioether radicals include methylthio, ethylthio,n-propylthio, isopropylthio, n-butylthio, iso-butylthio, sec-butylthio,tert-butylthio, methanesulfonyl, ethanesulfinyl, and the like.

The term “alkynyl,” as used herein, alone or in combination, refers to astraight-chain or branched chain hydrocarbon radical having one or moretriple bonds and containing from 2 to 20 carbon atoms. In certainembodiments, the alkynyl comprises from 2 to 6 carbon atoms. In furtherembodiments, the alkynyl comprises from 2 to 4 carbon atoms. The term“alkynylene” refers to a carbon-carbon triple bond attached at twopositions such as ethynylene (—C:::C—, —C≡C—). Examples of alkynylradicals include ethynyl, propynyl, hydroxypropynyl, butyn-1-yl,butyn-2-yl, pentyn-1-yl, 3-methylbutyn-1-yl, hexyn-2-yl, and the like.Unless otherwise specified, the term “alkynyl” may include “alkynylene”groups.

The terms “amido” and “carbamoyl” as used herein, alone or incombination, refer to an amino group as described below attached to theparent molecular moiety through a carbonyl group, or vice versa. Theterm “C-amido” as used herein, alone or in combination, refers to a—C(O)N(RR′) group with R and R′ as defined herein or as defined by thespecifically enumerated “R” groups designated. The term “N-amido” asused herein, alone or in combination, refers to a RC(O)N(R′)-group, withR and R′ as defined herein or as defined by the specifically enumerated“R” groups designated. The term “acylamino” as used herein, alone or incombination, embraces an acyl group attached to the parent moietythrough an amino group. An example of an “acylamino” group isacetylamino (CH₃C(O)NH—).

The term “amino,” as used herein, alone or in combination, refers to—NRR′, wherein R and R′ are independently selected from the groupconsisting of hydrogen, alkyl, acyl, heteroalkyl, aryl, cycloalkyl,heteroaryl, and heterocycloalkyl, any of which may themselves beoptionally substituted. Additionally, R and R′ may combine to formheterocycloalkyl, either of which may be optionally substituted.

The term “aryl,” as used herein, alone or in combination, means acarbocyclic aromatic system containing one, two or three rings whereinsuch polycyclic ring systems are fused together. The term “aryl”embraces aromatic groups such as phenyl, naphthyl, anthracenyl, andphenanthryl.

The term “arylalkenyl” or “aralkenyl,” as used herein, alone or incombination, refers to an aryl group attached to the parent molecularmoiety through an alkenyl group.

The term “arylalkoxy” or “aralkoxy,” as used herein, alone or incombination, refers to an aryl group attached to the parent molecularmoiety through an alkoxy group.

The term “arylalkyl” or “aralkyl,” as used herein, alone or incombination, refers to an aryl group attached to the parent molecularmoiety through an alkyl group.

The term “arylalkynyl” or “aralkynyl,” as used herein, alone or incombination, refers to an aryl group attached to the parent molecularmoiety through an alkynyl group.

The term “arylalkanoyl” or “aralkanoyl” or “aroyl,” as used herein,alone or in combination, refers to an acyl radical derived from anaryl-substituted alkanecarboxylic acid such as benzoyl, napthoyl,phenylacetyl, 3-phenylpropionyl (hydrocinnamoyl), 4-phenylbutyryl,(2-naphthyl)acetyl, 4-chlorohydrocinnamoyl, and the like.

The term aryloxy as used herein, alone or in combination, refers to anaryl group attached to the parent molecular moiety through an oxy.

The terms “benzo” and “benz,” as used herein, alone or in combination,refer to the divalent radical C6H4═ derived from benzene. Examplesinclude benzothiophene and benzimidazole.

The term “carbamate,” as used herein, alone or in combination, refers toan ester of carbamic acid (—NHCOO—) which may be attached to the parentmolecular moiety from either the nitrogen or acid end, and which may beoptionally substituted as defined herein.

The term “O-carbamyl” as used herein, alone or in combination, refers toa —OC(O)NRR′, group-with R and R′ as defined herein.

The term “N-carbamyl” as used herein, alone or in combination, refers toa ROC(O)NR′-group, with R and R′ as defined herein.

The term “carbonyl,” as used herein, when alone includes formyl [—C(O)H]and in combination is a —C(O)— group.

The term “carboxyl” or “carboxy,” as used herein, refers to —C(O)OH orthe corresponding “carboxylate” anion, such as is in a carboxylic acidsalt. An “O-carboxy” group refers to a RC(O)O— group, where R is asdefined herein. A “C-carboxy” group refers to a —C(O)OR groups where Ris as defined herein.

The term “cyano,” as used herein, alone or in combination, refers to—CN.

The term “cycloalkyl,” or, alternatively, “carbocycle,” as used herein,alone or in combination, refers to a saturated or partially saturatedmonocyclic, bicyclic or tricyclic alkyl group wherein each cyclic moietycontains from 3 to 12 carbon atom ring members and which may optionallybe a benzo fused ring system which is optionally substituted as definedherein. In certain embodiments, the cycloalkyl will comprise from 5 to 7carbon atoms. Examples of such cycloalkyl groups include cyclopropyl,cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, tetrahydronapthyl,indanyl, octahydronaphthyl, 2,3-dihydro-1H-indenyl, adamantyl and thelike. “Bicyclic” and “tricyclic” as used herein are intended to includeboth fused ring systems, such as decahydronaphthalene,octahydronaphthalene as well as the multicyclic (multicentered)saturated or partially unsaturated type. The latter type of isomer isexemplified in general by, bicyclo[1,1,1]pentane, camphor, adamantane,and bicyclo[3,2,1]octane.

The term “ester,” as used herein, alone or in combination, refers to acarboxy group bridging two moieties linked at carbon atoms.

The term “ether,” as used herein, alone or in combination, refers to anoxy group bridging two moieties linked at carbon atoms.

The term “halo,” or “halogen,” as used herein, alone or in combination,refers to fluorine, chlorine, bromine, or iodine.

The term “haloalkoxy,” as used herein, alone or in combination, refersto a haloalkyl group attached to the parent molecular moiety through anoxygen atom.

The term “haloalkyl,” as used herein, alone or in combination, refers toan alkyl radical having the meaning as defined above wherein one or morehydrogens are replaced with a halogen. Specifically embraced aremonohaloalkyl, dihaloalkyl and polyhaloalkyl radicals. A monohaloalkylradical, for one example, may have an iodo, bromo, chloro or fluoro atomwithin the radical. Dihalo and polyhaloalkyl radicals may have two ormore of the same halo atoms or a combination of different halo radicals.Examples of haloalkyl radicals include fluoromethyl, difluoromethyl,trifluoromethyl, chloromethyl, dichloromethyl, trichloromethyl,pentafluoroethyl, heptafluoropropyl, difluorochloromethyl,dichlorofluoromethyl, difluoroethyl, difluoropropyl, dichloroethyl anddichloropropyl. “Haloalkylene” refers to a haloalkyl group attached attwo or more positions. Examples include fluoromethylene (—CFH—),difluoromethylene (—CF₂—), chloromethylene (—CHCl—) and the like.

The term “heteroalkyl,” as used herein, alone or in combination, refersto a stable straight or branched chain, or cyclic hydrocarbon radical,or combinations thereof, fully saturated or containing from 1 to 3degrees of unsaturation, consisting of the stated number of carbon atomsand from one to three heteroatoms selected from the group consisting ofO, N, and S, and wherein the nitrogen and sulfur atoms may optionally beoxidized and the nitrogen heteroatom may optionally be quaternized. Theheteroatom(s) O, N and S may be placed at any interior position of theheteroalkyl group. Up to two heteroatoms may be consecutive, such as,for example, —CH₂—NH—OCH_(3.)

The term “heteroaryl,” as used herein, alone or in combination, refersto a 3 to 15 membered unsaturated heteromonocyclic ring, or a fusedmonocyclic, bicyclic, or tricyclic ring system in which at least one ofthe fused rings is aromatic, which contains at least one atom selectedfrom the group consisting of O, S, and N. In certain embodiments, theheteroaryl will comprise from 5 to 7 carbon atoms. The term alsoembraces fused polycyclic groups wherein heterocyclic rings are fusedwith aryl rings, wherein heteroaryl rings are fused with otherheteroaryl rings, wherein heteroaryl rings arc fused withheterocycloalkyl rings, or wherein heteroaryl rings are fused withcycloalkyl rings. Examples of heteroaryl groups include pyrrolyl,pyrrolinyl, imidazolyl, pyrazolyl, pyridyl, pyrimidinyl, pyrazinyl,pyridazinyl, triazolyl, pyranyl, furyl, thienyl, oxazolyl, isoxazolyl,oxadiazolyl, thiazolyl, thiadiazolyl, isothiazolyl, indolyl, isoindolyl,indolizinyl, benzimidazolyl, quinolyl, isoquinolyl, quinoxalinyl,quinazolinyl, indazolyl, benzotriazolyl, benzodioxolyl, benzopyranyl,benzoxazolyl, benzoxadiazolyl, benzothiazolyl, benzothiadiazolyl,benzofuryl, benzothienyl, chromonyl, coumarinyl, benzopyranyl,tetrahydroquinolinyl, tetrazolopyridazinyl, tetrahydroisoquinolinyl,thienopyridinyl, furopyridinyl, pyrrolopyridinyl and the like. Exemplarytricyclic heterocyclic groups include carbazolyl, benzidolyl,phenanthrolinyl, dibenzofuranyl, acridinyl, phenanthridinyl, xanthenyland the like.

The terms “heterocycloalkyl” and, interchangeably, “heterocycle,” asused herein, alone or in combination, each refer to a saturated,partially unsaturated, or fully unsaturated monocyclic, bicyclic, ortricyclic heterocyclic group containing at least one heteroatom as aring member, wherein each the heteroatom may be independently selectedfrom the group consisting of nitrogen, oxygen, and sulfur In certainembodiments, the hetercycloalkyl will comprise from 1 to 4 heteroatomsas ring members. In further embodiments, the hetercycloalkyl willcomprise from 1 to 2 heteroatoms as ring members. In certainembodiments, the hetercycloalkyl will comprise from 3 to 8 ring membersin each ring. In further embodiments, the hetercycloalkyl will comprisefrom 3 to 7 ring members in each ring. In yet further embodiments, thehetercycloalkyl will comprise from 5 to 6 ring members in each ring.“Heterocycloalkyl” and “heterocycle” are intended to include sulfones,sulfoxides, N-oxides of tertiary nitrogen ring members, and carbocyclicfused and benzo fused ring systems; additionally, both terms alsoinclude systems where a heterocycle ring is fused to an aryl group, asdefined herein, or an additional heterocycle group. Examples ofheterocycle groups include aziridinyl, azetidinyl, 1,3-benzodioxolyl,dihydroisoindolyl, dihydroisoquinolinyl, dihydrocinnolinyl,dihydrobenzodioxinyl, dihydro[1,3]oxazolo[4,5-b]pyridinyl,benzothiazolyl, dihydroindolyl, dihy-dropyridinyl, 1,3-dioxanyl,1,4-dioxanyl, 1,3-dioxolanyl, isoindolinyl, morpholinyl, piperazinyl,pyrrolidinyl, tetrahydropyridinyl, piperidinyl, thiomorpholinyl, and thelike. The heterocycle groups may be optionally substituted unlessspecifically prohibited.

The term “hydrazinyl” as used herein, alone or in combination, refers totwo amino groups joined by a single bond, i.c., —N—N—.

The term “hydroxy,” as used herein, alone or in combination, refers to—OH.

The term “hydroxyalkyl,” as used herein, alone or in combination, refersto a hydroxy group attached to the parent molecular moiety through analkyl group.

The term “imino,” as used herein, alone or in combination, refers to═N—.

The term “iminohydroxy,” as used herein, alone or in combination, refersto ═N(OH) and ═N—O—.

The phrase “in the main chain” refers to the longest contiguous oradjacent chain of carbon atoms starting at the point of attachment of agroup to the compounds of any one of the formulas disclosed herein.

The term “isocyanato” refers to a —NCO group.

The term “isothiocyanato” refers to a —NCS group.

The phrase “linear chain of atoms” refers to the longest straight chainof atoms independently selected from carbon, nitrogen, oxygen andsulfur.

The term “lower,” as used herein, alone or in a combination, where nototherwise specifically defined, means containing from 1 to and including6 carbon atoms.

The term “lower aryl,” as used herein, alone or in combination, meansphenyl or naphthyl, either of which may be optionally substituted asprovided.

The term “lower heteroaryl,” as used herein, alone or in combination,means either 1) monocyclic heteroaryl comprising five or six ringmembers, of which between one and four the members may be heteroatomsselected from the group consisting of O, S, and N, or 2) bicyclicheteroaryl, wherein each of the fused rings comprises five or six ringmembers, comprising between them one to four heteroatoms selected fromthe group consisting of O, S, and N.

The term “lower cycloalkyl,” as used herein, alone or in combination,means a monocyclic cycloalkyl having between three and six ring members.Lower cycloalkyls may be unsaturated. Examples of lower cycloalkylinclude cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl.

The term “lower heterocycloalkyl,” as used herein, alone or incombination, means a monocyclic heterocycloalkyl having between threeand six ring members, of which between one and four may be heteroatomsselected from the group consisting of O, S, and N. Examples of lowerheterocycloalkyls include pyrrolidinyl, imidazolidinyl, pyrazolidinyl,piperidinyl, piperazinyl, and morpholinyl. Lower heterocycloalkyls maybe unsaturated.

The term “lower amino,” as used herein, alone or in combination, refersto —NRR′, wherein R and R′ are independently selected from the groupconsisting of hydrogen, lower alkyl, and lower heteroalkyl, any of whichmay be optionally substituted. Additionally, the R and R′ of a loweramino group may combine to form a five-or six-membered heterocycloalkyl,either of which may be optionally substituted.

The term “mercaptyl” as used herein, alone or in combination, refers toan RSgroup, where R is as defined herein.

The term “nitro,” as used herein, alone or in combination, refers to—NO2.

The terms “oxy” or “oxa,” as used herein, alone or in combination, referto —O—.

The term “oxo,” as used herein, alone or in combination, refers to ═O.

The term “perhaloalkoxy” refers to an alkoxy group where all of thehydrogen atoms are replaced by halogen atoms.

The term “perhaloalkyl” as used herein, alone or in combination, refersto an alkyl group where all of the hydrogen atoms are replaced byhalogen atoms.

The terms “sulfonate,” “sulfonic acid,” and “sulfonic,” as used herein,alone or in combination, refer the —SO₃H group and its anion as thesulfonic acid is used in salt formation.

The term “sulfanyl,” as used herein, alone or in combination, refers to—S—.

The term “sulfinyl,” as used herein, alone or in combination, refers to—S(O)—.

The term “sulfonyl,” as used herein, alone or in combination, refers to—S(O)₂—.

The term “N-sulfonamido” refers to a RS(═O)₂NR′— group with R and R′ asdefined herein.

The term “S-sulfonamido” refers to a —S(═O)₂NRR′, group, with R and R′as defined herein.

The terms “thia” and “thio,” as used herein, alone or in combination,refer to a —S— group or an ether wherein the oxygen is replaced withsulfur. The oxidized derivatives of the thio group, namely sulfinyl andsulfonyl, are included in the definition of thia and thio.

The term “thiol,” as used herein, alone or in combination, refers to an—SH group.

The term “thiocarbonyl,” as used herein, when alone includes thioformyl—C(S)H and in combination is a —C(S)— group.

The term “N-thiocarbamyl” refers to an ROC(S)NR′— group, with R and R′as defined herein.

The term “O-thiocarbamyl” refers to a —OC(S)NRR′, group with R and R′ asdefined herein.

The term “thiocyanato” refers to a —CNS group.

The term “trihalomethanesulfonamido” refers to a X₃CS(O)₂NR— group withX is a halogen and R as defined herein.

The term “trihalomethanesulfonyl” refers to a X₃CS(O)₂— group where X isa halogen.

The term “trihalomethoxy” refers to a X₃CO— group where X is a halogen.

The term “trisubstituted silyl,” as used herein, alone or incombination, refers to a silicone group substituted at its three freevalences with groups as listed herein under the definition ofsubstituted amino. Examples include trimethysilyl,tert-butyldimethylsilyl, triphenylsilyl and the like.

Any definition herein may be used in combination with any otherdefinition to describe a composite structural group. By convention, thetrailing element of any such definition is that which attaches to theparent moiety. For example, the composite group alkylamido wouldrepresent an alkyl group attached to the parent molecule through anamido group, and the term alkoxyalkyl would represent an alkoxy groupattached to the parent molecule through an alkyl group.

When a group is defined to be “null,” what is meant is that the group isabsent.

The term “optionally substituted” means the anteceding group may besubstituted or unsubstituted. When substituted, the substituents of an“optionally substituted” group may include, without limitation, one ormore substituents independently selected from the following groups or aparticular designated set of groups, alone or in combination: loweralkyl, lower alkenyl, lower alkynyl, lower alkanoyl, lower heteroalkyl,lower heterocycloalkyl, lower haloalkyl, lower haloalkenyl, lowerhaloalkynyl, lower perhaloalkyl, lower perhaloalkoxy, lower cycloalkyl,phenyl, aryl, aryloxy, lower alkoxy, lower haloalkoxy, oxo, loweracyloxy, carbonyl, carboxyl, lower alkylcarbonyl, lower carboxyester,lower carboxamido, cyano, hydrogen, halogen, hydroxy, amino, loweralkylamino, arylamino, amido, nitro, thiol, lower alkylthio, lowerhaloalkylthio, lower perhaloalkylthio, arylthio, sulfonate, sulfonicacid, trisubstituted silyl, N₃, SH, SCH₃, C(O)CH₃, CO₂CH₃, CO₂H,pyridinyl, thiophene, furanyl, lower carbamate, and lower urea. Twosubstituents may be joined together to form a fused five—, six—, orseven-membered carbocyclic or heterocyclic ring consisting of zero tothree heteroatoms, for example forming methylenedioxy or ethylenedioxy.An optionally substituted group may be unsubstituted (e.g., —CH₂CH₃),fully substituted (e.g., —CF₂CF₃), monosubstituted (e.g., —CH₂CH₂F) orsubstituted at a level anywhere in-between fully substituted andmonosubstituted (e.g., —CH₂CF₃). Where substituents are recited withoutqualification as to substitution, both substituted and unsubstitutedforms are encompassed. Where a substituent is qualified as“substituted,” the substituted form is specifically intended.Additionally, different sets of optional substituents to a particularmoiety may be defined as needed; in these cases, the optionalsubstitution will be as defined, often immediately following the phrase,“optionally substituted with.”

The term R or the term R′, appearing by itself and without a numberdesignation, unless otherwise defined, refers to a moiety selected fromthe group consisting of hydrogen, alkyl, cycloalkyl, heteroalkyl, aryl,heteroaryl and heterocycloalkyl, any of which may be optionallysubstituted. Such R and R′ groups should be understood to be optionallysubstituted as defined herein. Whether an R group has a numberdesignation or not, every R group, including R, R′ and R″ where n=(1, 2,3, . . . n), every substituent, and every term should be understood tobe independent of every other in terms of selection from a group. Shouldany variable, substituent, or term (e.g. aryl, heterocycle, R, etc.)occur more than one time in a formula or generic structure, itsdefinition at each occurrence is independent of the definition at everyother occurrence. Those of skill in the art will further recognize thatcertain groups may be attached to a parent molecule or may occupy aposition in a chain of elements from either end as written. Thus, by wayof example only, an unsymmetrical group such as —C(O)N(R)— may beattached to the parent moiety at either the carbon or the nitrogen.

(AA) is polypeptide chain including one or more natural orunnaturalα-amino acids linked together by peptide bonds or natural orunnatural β-amino acid(s), linked together by peptide bonds orcombination of α- and β-amino acid(s), linked by peptide bonds. Thepolypeptide chain (AA) may be a homopolypeptide chain or aheteropolypeptide chain, and may be any appropriate length. Forinstance, in some embodiments, the polypeptide chain may include 1 to100 α-amino acid(s), 1 to 90 α-amino acid(s), 1 to 80 α-amino acid(s), 1to 70 α-amino acid(s), 1 to 60 α-amino acid(s), 1 to 50 α-amino acid(s),1 to 40 α-amino acid(s), 1 to 30 α-amino acid(s), 1 to 20 α-aminoacid(s), or even 1 to 10 α-amino acid(s), In some embodiments, theα-amino acids of the polypeptide chain (AA) are selected from the groupconsisting of aspartic acid, asparagine, arginine, histidine, lysine,glutamic acid, glutamine, serine, and homoserine. In some embodiments,the α-amino acids of the polypeptide chain (AA) are selected from thegroup consisting of aspartic acid, glutamic acid, serine, andhomoserine. In some embodiments, the polypeptide chain (AA) refers to asingle amino (e.g., either aspartic acid or serine). In someembodiments, the polypeptide chain may include 1 to 100 β-amino acid(s),1 to 90 β-amino acid(s), 1 to 80 β-amino acid(s), 1 to 70 β-aminoacid(s), 1 to 60 β-amino acid(s), 1 to 50 β-amino acid(s), 1 to 40β-amino acid(s), 1 to 30 β-amino acid(s), 1 to 20 β-amino acid(s), oreven 1 to 10 β-amino acid(s). In some embodiments, a combination ofα-amino acids and β-amino acids of the polypeptide chain may beincluded.

Asymmetric centers exist in the compounds disclosed herein. Thesecenters are designated by the symbols “R” or “S,” depending on theconfiguration of substituents around the chiral carbon atom. It shouldbe understood that the disclosure encompasses all stereochemicalisomeric forms, including diastereomeric, enantiomeric, and epimericforms, as well as d-isomers and 1-isomers, and mixtures thereof.Individual stereoisomers of compounds can be prepared synthetically fromcommercially available starting materials which contain chiral centersor by preparation of mixtures of enantiomeric products followed byseparation such as conversion to a mixture of diastereomers followed byseparation or recrystallization, chromatographic techniques, directseparation of enantiomers on chiral chromatographic columns, or anyother appropriate method known in the art. Starting compounds ofparticular stereochemistry are either commercially available or can bemade and resolved by techniques known in the art. Additionally, thecompounds disclosed herein may exist as geometric isomers. The presentdisclosure includes all cis, trans, syn, anti, entgegen (E), andzusammen (Z) isomers as well as the appropriate mixtures thereof.Additionally, compounds may exist as tautomers; all tautomeric isomersare provided by this disclosure. Additionally, the compounds disclosedherein can exist in unsolvated as well as solvated forms withpharmaceutically acceptable solvents such as water, ethanol, and thelike. In general, the solvated forms are considered equivalent to theunsolvated forms.

The term “bond” refers to a covalent linkage between two atoms, or twomoieties when the atoms joined by the bond are considered part of largersubstructure. A bond may be single, double, or triple unless otherwisespecified. A dashed line between two atoms in a drawing of a moleculeindicates that an additional bond may be present or absent at thatposition.

The term “disease” as used herein is intended to be generallysynonymous, and is used interchangeably with, the terms “disorder,”“syndrome,” and “condition” (as in medical condition), in that allreflect an abnormal condition of the human or animal body or of one ofits parts that impairs normal functioning, is typically manifested bydistinguishing signs and symptoms, and causes the human or animal tohave a reduced duration or quality of life.

The phrase “therapeutically effective” is intended to qualify the amountof active ingredients used in the treatment of a disease or disorder oron the effecting of a clinical endpoint.

The term “angiography” refers to a medical imaging technique forvisualizing the lumen of a blood vessel, for example, an artery.

The term “vasculature” refers to blood vessels, such as veins andarteries, in the body or in an organ or body part. Although portions ofthis disclosure may refer to veins and arteries, this disclosure shallbe applicable to any type of blood vessel within the “vascular system.”

The term “pulmonary and cardiac vasculature” as used herein includes allof the blood vessels within the lungs and/or heart, the chambers of theheart, the passages between the chambers of the heart, as well as theblood vessels between the lungs and heart, and blood vessels between thelungs or heart and other tissues and/or organs. The pulmonary andcardiac vasculature includes, but is not limited to, the pulmonary veinsand arteries and associated capillaries, the left and right atria of theheart, the left and right ventricles of the heart, the myocardium, theaorta and aortic arch, the coronary artery, the coronary arteries, thesubclavian arteries, and the carotid arteries.

The term “abnormality” refers to the presence of an activity or featurewhich differs from a normal activity or feature. An abnormality mayrefer to a disease or disorder in need of treatment.

The term “stenosis” is defined to be a narrowing in a blood vessel orother tubular organ or structure, i.e. a vasoconstricting condition.This term also encompasses terms such as “restenosis” and “in-stentrestenosis”. The term “restenosis” refers to the re-occurence ofstenosis.

The term “occlusion” as used herein refers to an obstruction or closureof a passageway or vessel.

The term “aneurysm” refers to a localized dilation of a blood vessel,particularly of the aorta or a peripheral artery. Aneurysms are relatedto arteriosclerosis, cystic medial necrosis, pathogen infection,aortitis, and trauma, each of which may contribute to weakening of thevessel wall. Common aneurysm sites include the abdominal aorta, thethoracic aorta, peripheral arteries such as the popliteal, iliac, andfemoral arteries.

The phrase “short-wavelength dye” in the context of this invention isintended to mean a dye capable of absorbing and/or emitting light in theblue or green portion of the electromagnetic spectrum.

The phrase “long-wavelength dye” in the context of this invention isintended to mean a dye capable of absorbing and/or emitting light in thered or infrared portion of the electromagnetic spectrum.

The term “low molecular weight dye” refers to a dye having a molecularweight of less than about 1,000 grams per mole (“g/mol”).

The term “high molecular weight dye” refers to a dye having a molecularweight of greater than about 1,000 grams per mole (“g/mol”).

The term “therapeutically acceptable” refers to those compounds (orsalts, prodrugs, tautomers, zwitterionic forms, etc.) which are suitablefor use in contact with the tissues of patients without undue toxicity,irritation, and allergic response, are commensurate with a reasonablebenefit/risk ratio, and are effective for their intended use.

As used herein, reference to “treatment” of a patient is intended toinclude prophylaxis. Treatment may also be preemptive in nature, i.e.,it may include prevention of disease. Prevention of a disease mayinvolve complete protection from disease, for example as in the case ofprevention of infection with a pathogen, or may involve prevention ofdisease progression. For example, prevention of a disease may not meancomplete foreclosure of any effect related to the diseases at any level,but instead may mean prevention of the symptoms of a disease to aclinically significant or detectable level. Prevention of diseases mayalso mean prevention of progression of a disease to a later stage of thedisease.

The term “patient” is generally synonymous with the term “subject” andincludes all mammals including humans. Examples of patients includehumans, livestock such as cows, goats, sheep, pigs, and rabbits, andcompanion animals such as dogs, cats, rabbits, and horses.

The term “prodrug” refers to a compound that is made more active invivo. Certain compounds disclosed herein may also exist as prodrugs, asdescribed in Hydrolysis in Drug and Prodrug Metabolism: Chemistry,Biochemistry, and Enzymology (Testa, Bernard and Mayer, Joachim M.Wiley-VHCA, Zurich, Switzerland 2003). Prodrugs of the compoundsdescribed herein are structurally modified forms of the compound thatreadily undergo chemical changes under physiological conditions toprovide the compound. Additionally, prodrugs can be converted to thecompound by chemical or biochemical methods in an ex vivo environment.For example, prodrugs can be slowly converted to a compound when placedin a transdermal patch reservoir with a suitable enzyme or chemicalreagent. Prodrugs are often useful because, in some situations, they maybe easier to administer than the compound, or parent drug. They may, forinstance, be bioavailable by oral administration whereas the parent drugis not. The prodrug may also have improved solubility in pharmaceuticalcompositions over the parent drug. A wide variety of prodrug derivativesare known in the art, such as those that rely on hydrolytic cleavage oroxidative activation of the prodrug. An example, without limitation, ofa prodrug would be a compound which is administered as an ester (the“prodrug”), but then is metabolically hydrolyzed to the carboxylic acid,the active entity. Additional examples include peptidyl derivatives of acompound.

The compounds disclosed herein can exist as therapeutically acceptablesalts. The present disclosure includes compounds listed above in theform of salts, including acid addition salts. Suitable salts includethose formed with both organic and inorganic acids. Such acid additionsalts will normally be pharmaceutically acceptable. However, salts ofnon-pharmaceutically acceptable salts may be of utility in thepreparation and purification of the compound in question. Basic additionsalts may also be formed and be pharmaceutically acceptable. For a morecomplete discussion of the preparation and selection of salts, refer toPharmaceutical Salts: Properties, Selection, and Use (Stahl, P.Heinrich. Wiley-VCHA, Zurich, Switzerland, 2002).

The term “therapeutically acceptable salt,” as used herein, representssalts or zwitterionic forms of the compounds disclosed herein which arewater or oil-soluble or dispersible and therapeutically acceptable asdefined herein. The salts can be prepared during the final isolation andpurification of the compounds or separately by reacting the appropriatecompound in the form of the free base with a suitable acid.Representative acid addition salts include acetate, adipate, alginate,L-ascorbate, aspartate, benzoate, benzenesulfonate (besylate),bisulfate, butyrate, camphorate, camphorsulfonate, citrate, digluconate,formate, fumarate, gentisate, glutarate, glycerophosphate, glycolate,hemisulfate, heptanoate, hexanoate, hippurate, hydrochloride,hydrobromide, hydroiodide, 2-hydroxyethansulfonate (isethionate),lactate, maleate, malonate, DL-mandelate, mesitylenesulfonate,methanesulfonate, naphthylenesulfonate, nicotinate,2-naphthalenesulfonate, oxalate, pamoate, pectinate, persulfate,3-phenylproprionate, phosphonate, picrate, pivalate, propionate,pyroglutamate, succinate, sulfonate, tartrate, L-tartrate,trichloroacetate, trifluoroacetate, phosphate, glutamate, bicarbonate,para-toluenesulfonate (p-tosylate), and undecanoate. Also, basic groupsin the compounds disclosed herein can be quaternized with methyl, ethyl,propyl, and butyl chlorides, bromides, and iodides; dimethyl, diethyl,dibutyl, and diamyl sulfates; decyl, lauryl, myristyl, and sterylchlorides, bromides, and iodides; and benzyl and phenethyl bromides.Examples of acids which can be employed to form therapeuticallyacceptable addition salts include inorganic acids such as hydrochloric,hydrobromic, sulfuric, and phosphoric, and organic acids such as oxalic,maleic, succinic, and citric. Salts can also be formed by coordinationof the compounds with an alkali metal or alkaline earth ion. Hence, thepresent disclosure contemplates sodium, potassium, magnesium, andcalcium salts of the compounds disclosed herein, and the like.

Basic addition salts can be prepared during the final isolation andpurification of the compounds by reacting a carboxy group with asuitable base such as the hydroxide, carbonate, or bicarbonate of ametal cation or with ammonia or an organic primary, secondary, ortertiary amine. The cations of therapeutically acceptable salts includelithium, sodium, potassium, calcium, magnesium, and aluminum, as well asnontoxic quaternary amine cations such as ammonium, tetramethylammonium,tetraethylammonium, methylamine, dimethylamine, trimethylamine,triethylamine, diethylamine, ethylamine, tributylamine, pyridine,N,N-dimethylaniline, N-methylpiperidine, N-methylmorpholine,dicyclohexylamine, procaine, dibenzylamine, N,N-dibenzylphenethylamine,1-ephenamine, and N,N′-dibenzylethylenediamine. Other representativeorganic amines useful for the formation of base addition salts includeethylenediamine, ethanolamine, diethanolamine, piperidine, andpiperazine.

A salt of a compound can be made by reacting the appropriate compound inthe form of the free base with the appropriate acid.

Compositions

The present disclosure provides a composition for assessing eyevasculature in a subject in need thereof, comprising a fluorescent dye.The fluorescent dyes of the present disclosure tend to have absorption,excitation, and emission wavelengths that are all within thenear-infrared (NIR) or visible spectrum of about 350 nm or greater. Thisis beneficial for diagnostic procedures since visible and NIR light arenot likely to damage tissue. In contrast, ultraviolet (UV) light thathas a wavelength of less than about 350 nm can damage tissue. Lighthaving a wavelength of about 350 nm or greater tends to penetrate intotissues thereby permitting diagnostic procedures to be conducted intissues of interest that may not be reachable using UV wavelengths thatare less than about 350 nm.

Synthesis of pyrazine derivatives, in general, have been previouslystudied and described. Preparation procedures for some of the pyrazinederivatives of the present invention, using procedures similar to theabove references, are described later in Examples 2 and 4-18.

Pyrazine dyes of the invention may be characterized as demonstratingabsorption in the visible region and emission/fluorescence in thevisible or near-infrared region, tend to exhibit significant Stokesshifts, and tend to be cleared from the body via the kidneys. Theseproperties allow flexibility in both tuning a molecule to a desiredwavelength and introducing a variety of substituents to improvepharmacokinetic (PK) and pharmacodynamics (PD) properties. The pyrazinederivatives described herein may be designed to be hydrophilic and/orhave rigid functionality, thus providing desired pharmacokineticproperties appropriate for angiography in general and ocular angiographyin particular.

Provided are compositions comprising a compound of structural Formula I

or a salt thereof; wherein

-   X¹ and X² are independently chosen from —CO(AA), —CN, —CO₂R¹,    CONR²R³, —COR⁴, —NO₂, —SOR³⁵, —SO₂R⁶, —SO₂R⁷ and —PO₃R⁸R⁹;-   Y¹ and Y² atr independently chosen from —OR¹⁰, —SR¹¹, —NR¹²R¹³,    —N(R¹⁴)COR¹⁵, CONH(PS); —P(R¹⁶)₂, —P(OR¹⁷)₂ and

-   each Z¹ is independently chosen from a bond, —CR¹⁸R¹⁹—, —O—, —NR²⁰—,    —NCOR²¹—, —S—, —SO—, and —SO₂—;-   each R¹ to R²¹ are independently chosen from hydrogen, C₁-C₁₀ alkyl    optionally substituted with hydroxyl and carboxylic acid, C₃-C₆    polyhydroxylated alkyl, C₅-C₁₀ aryl, C₅-C₁₀ heteroaryl, C₃-C₅    heterocycloalkyl optionally substituted with C(O), —(CH₂)_(a)CO₂H    optionally substituted with C₅-C₁₀ heteroaryl, —(CH₂)_(a)CONR³⁰R³¹,    —(CH₂)_(a)NHSO₃ ⁻, —(CH₂)_(a)NHSO₃H, —(CH₂)_(a)OH, —(CH₂)_(a)OPO₃ ⁼,    —(CH₂)_(a)OPO₃H₂, —(CH₂)_(a)OPO₃H⁻, —(CH₂)_(a)OR²², —(CH₂)_(a) OSO₃    ⁻, —(CH₂)_(a)OSO₃H, —(CH₂)_(a)PO₃ ⁼, —(CH₂)_(a)PO₃H₂,    —(CH₂)_(a)PO₃H⁻, —(CH₂)_(a)SO₃ ⁻, —(CH₂)_(a)SO₃H,    —(CH₂)_(d)CO(CH₂CH₂O)_(c)R²³, —(CH₂)_(d)(CH₂CH₂O)_(c)R²⁴,    —(CHCO₂H)_(a)CO₂H, —CH₂(CHNH₂)_(a)CH₂NR²⁵R²⁶ —CH₂(CHOH)_(a)CO₂H,    —CH₂(CHOH)_(a)R²⁷, —CH[(CH₂)_(b)NH₂]_(a)CH₂OH,    —CH[(CH₂)_(b)NH₂]_(a)CO₂H, and —(CH₂)_(a)NR²⁸R²⁹;-   each R²² to R³¹ are independently chosen from hydrogen, C₁-C₁₀    alkyl, and C₁-C₅-dicarboxylic acid;

R³⁵ is chosen from C₁-C₁₀ alkyl optionally substituted with hydroxyl andcarboxylic acid, C₃-C₆ polyhydroxylated alkyl, C₅-C₁₀ aryl, C₅-C₁₀heteroaryl, C₃-C₅ heterocycloalkyl optionally substituted with C(O),—(CH₂)_(a)CO₂H optionally substituted with C₅-C₁₀ heteroaryl,—(CH₂)_(a)CONR³⁰R³¹, —(CH₂)_(a)NHSO₃ ⁻, —(CH₂)_(a)NHSO₃H, —(CH₂)_(a)OH,—(CH₂)_(a)OPO₃ ⁻, —(CH₂)_(a)OPO₃H₂, —(CH₂)_(a)OPO₃H⁻, —(CH₂)_(a)OR²²,—(CH₂)_(a)OSO₃ ⁻, —(CH₂)_(a)OS03H, —(CH₂)_(a)PO₃ ⁼, —(CH₂)_(a)PO₃H₂,—(CH₂)_(a)PO₃H⁻, —(CH₂)_(a)SO₃ ⁻, —(CH₂)_(a)SO₃H,—(CH₂)_(d)CO(CH₂CH₂O)_(c)R²³, —(CH₂)_(d)(CH₂CH₂O)_(c)R²⁴,—(CHCO₂H)_(a)CO₂H, —CH₂(CHNH₂)_(a)CH₂NR²⁵R²⁶, —CH₂(CHOH)_(a)CO₂H,—CH₂(CHOH)_(a)R²⁷, —CH[(CH₂)_(b)NH₂]_(a)CH₂OH,—CH[(CH₂)_(b)NH₂]_(a)CO₂H, and —(CH₂)_(a)NR²⁸R²⁹;

-   (AA) is a polypeptide chain comprising one or more natural or    unnatural β-amino acids or γ-amino acids linked together by peptide    bonds;-   (PS) is a sulfated or non-sulfated polysaccharide chain comprising    one or more monosaccharide units connected by glycosidic linkages;    and-   each ‘a’, ‘b’, and ‘d’ are independently chosen from 0 to 10, ‘c’ is    chosen from 1 to 100 and each of ‘m’ and ‘n’ independently is an    integer from 1 to 3.

In an embodiment, said compounds are at least partially renallyexcretable.

In an embodiment, said compounds are completely renally excretable.

Methods

In these methods, an effective amount of a pyrazine derivative isadministered into the body of a patient (e.g., a mammal such as a humanor animal subject). An “effective amount” herein generally refers to anamount of pyrazine derivative that is sufficient to enable angiographicfunction to be analyzed. The pyrazine derivative in the body of thepatient is exposed to at least one of visible and near infrared light.Due to this exposure of the pyrazine derivative to the visible and/orinfrared light, the pyrazine derivative emanates spectral energy thatmay be detected by appropriate detection equipment. This spectral energyemanating from the pyrazine derivative may be detected using anappropriate detection mechanism such as an invasive or non-invasiveoptical probe or photographic equipment such as a fundus camera. Herein,“emanating” or the like refers to spectral energy that is emitted and/orfluoresced from a pyrazine derivative. Angiographic function can bedetermined based the spectral energy that is detected. For example, aninitial amount of the amount of pyrazine derivative present in the bodyof a patient may be determined by a magnitude/intensity of lightemanated from the pyrazine derivative that is detected (e.g., in theretina). As the pyrazine derivative is cleared from the body, themagnitude/intensity of detected light generally diminishes. Accordingly,a rate at which this magnitude of detected light diminishes may becorrelated with a subject's vasculature. This detection may be doneperiodically or in substantially real time.

The present disclosure provides new methods for visualizing thevasculature of a subject in need thereof, comprising the steps of:administering an effective amount of a compound of structural Formula I

or a salt thereof; wherein

-   X¹ and X² are independently chosen from —CO(AA), —CN, —CO₂R¹,    —CONR²R³, —COR⁴, —NO₂, —SOR³⁵, —SO₂R⁶, —SO₂R⁷ and —PO₃R⁸R⁹;-   Y¹ and Y² are independently chosen from OR¹⁰, —SR¹¹, —NR¹²R¹³,    —N(R¹⁴)COR¹⁵, —CONH(PS); —P(R¹⁶)₂, —P(OR¹⁷)₂ and

-   each Z¹ is independently chosen from a bond, —CR¹⁸R¹⁹, —O—, —NR²⁰—,    —NCOR²¹—, —S—, —SO—, and S—O₂—;-   each R¹ to R²¹ are independently chosen from hydrogen, C₁-C₁₀ alkyl    optionally substituted with hydroxyl and carboxylic acid, C₃-C₆    polyhydroxylated alkyl, C₅-C₁₀ aryl, C₅-C₁₀ heteroaryl, C₃-C₅    heterocycloalkyl optionally substituted with C(O), —(CH₂)_(a)CO₂H    optionally substituted with C₅-C₁₀ heteroaryl, —(CH₂)_(a)CONR³⁰R³¹,    —(CH₂)_(a)NHSO₃ ⁻, —(CH₂)_(a)NHSO₃H, —(CH₂)_(a)OH, —(CH₂)_(a)OPO₃ ⁼,    —(CH₂)_(a)OPO₃H₂, —(CH₂)_(a)OPO₃H⁻, —(CH₂)_(a)OR²², —(CH₂)_(a)OSO₃    ⁻, —(CH₂)_(a)OSO₃H, —(CH₂)_(a)PO₃ ⁼, —(CH₂)_(a)PO₃H₂,    —(CH₂)_(a)PO₃H⁻, —(CH₂)_(a)SO₃ ⁻, —(CH₂)_(a)SO₃H,    —(CH₂)_(d)CO(CH₂CH₂O)_(c)R²³, —(CH₂)_(d)(CH₂CH₂O)_(c)R²⁴,    —(CHCO₂H)_(a)CO₂H, —CH₂(CHNH₂)_(a)CH₂NR²⁵R²⁶, —CH₂(CHOH)_(a)CO₂H,    —CH₂(CHOH)_(a)R²⁷, —CH[(CH₂)_(b)NH₂]_(a)CH₂OH,    —CH[(CH₂)_(b)NH₂]_(a)CO₂H, and —(CH₂)_(a)NR²⁸R²⁹;-   each R²² to R³¹ are independently chosen from hydrogen, C₁-C₁₀    alkyl, and C₁-C₅-dicarboxylic acid;-   R³⁵ is chosen from C₁-C₁₀ alkyl optionally substituted with hydroxyl    and carboxylic acid, C₃-C₆ polyhydroxylated alkyl, C₅-C₁₀ aryl,    C₅-C₁₀ heteroaryl, C₃-C₅ heterocycloalkyl optionally substituted    with C(O), —(CH₂)_(a)CO₂H optionally substituted with C₅-C₁₀    heteroaryl, —(CH₂)_(a)CONR³⁰R³¹, —(CH₂)_(a)NHSO₃ ⁻,    —(CH₂)_(a)NHSO₃H, —(CH₂)_(a)OH, —(CH₂)_(a)OPO₃ ⁼, —(CH₂)_(a)OPO₃H₂,    —(CH₂)_(a)OPO₃H⁻, —(CH₂)_(a)OR²², —(CH₂)_(a)OSO₃ ⁻, —(CH₂)_(a)OSO₃H,    —(CH₂)_(a)PO₃ ⁼, —(CH₂)_(a)PO₃H₂, —(CH₂)_(a)PO₃H⁻, —(CH₂)_(a)SO₃ ⁻,    —(CH₂)_(a)SO₃H, —(CH₂)_(d)CO(CH₂CH₂O)_(c)R²³,    —(CH₂)_(d)(CH₂CH₂O)_(c)R²⁴, —(CHCO₂H)_(a)CO₂H,    —CH₂(CHNH₂)_(a)CH₂NR²⁵R²⁶, —CH₂(CHOH)_(a)CO₂H, —CH₂(CHOH)_(a)R²⁷,    —CH[(CH₂)_(b)NH₂]_(a)CH₂OH, —CH[(CH₂)_(b)NH₂]_(a)CO₂H, and    —(CH₂)_(a)NR²⁸R²⁹;-   (AA) is a polypeptide chain comprising one or more natural or    unnatural amino acids linked together by peptide bonds;-   (PS) is a sulfated or non-sulfated polysaccharide chain comprising    one or more monosaccharide units connected by glycosidic linkages;    and-   each ‘a’, ‘b’, and ‘d’ are independently chosen from 0 to 10, ‘c’ is    chosen from 1 to 100 and each of ‘m’ and ‘n’ independently is an    integer from 1 to 3;    irradiating the subject's vasculature with non-ionizing radiation,    wherein the radiation causes the compound to fluoresce;    detecting the fluorescence of the compound in the subject's    vasculature; and    visualizing the vasculature within the subject based on the detected    fluorescence.

In an embodiment, said amino acids are chosen from α-amino acids,β-amino acids, and γ-amino acids.

In certain embodiments, the composition is administered intravenously.

In certain embodiments, the non-ionizing radiation has a wavelength ofat least 350 nm.

In certain embodiments, the detected fluorescence of the compound in thesubject's vasculature is measured over time.

In certain embodiments, the subject's pulmonary and cardiac vasculatureis visualized. In some embodiments, visualizing the subject's pulmonaryand cardiac vasculature comprises identifying abnormalities chosen fromstenosis, occlusions, aneurysms, and combinations thereof. In someembodiments, visualizing the subject's pulmonary and cardiac vasculaturecomprises comparing the detected fluorescence in the subject's pulmonaryand cardiac vasculature to that of normal pulmonary and cardiacvasculature under similar conditions.

In certain embodiments, the subject's eye vasculature is visualized. Insome embodiments, visualizing the subject's eye vasculature comprisesidentifying ocular abnormalities. In some embodiments, the ocular abnotialities are chosen from blood vessel architecture, ischemic spots,choroidal infarcts, Elschnig's spots, exudates, hemorrhages, andcombinations thereof. In some embodiments, the ocular abnormalities arechosen from vessel crossovers, vessel tortuosity, evidence of exudates,and combinations thereof. In some embodiments, visualizing the subject'seye vasculature comprises comparing the detected fluorescence in thesubject's eye to that of a normal eye under similar conditions.

The present disclosure provides new methods for assessing the locationof a disease or an injury in a subject's vasculature, comprising thesteps of: administering an effective amount of a compound of structuralFormula I

or a salt thereof; wherein

-   X¹ and X² are independently chosen from —CO(AA), —CN, —CO₂R¹,    —CONR²R³, —COR⁴, —NO₂, —SOR³⁵, —SO₂R⁶, —SO₂OR⁷ and —PO₃R⁸R⁹;-   Y¹ and Y² are independently chosen from —OR¹⁰, —SR¹¹, —NR¹²R¹³,    —N(R¹⁴)COR¹⁵, —CONH(PS); —P(R¹⁶)₂, —P(OR¹⁷)₂ and

-   each Z¹ is independently chosen from a bond, —CR¹⁸R¹⁹, —O—, —NR²⁰—,    —NCOR²¹—, —S—, —SO—, and —SO₂—;-   each R¹ to R²¹ are independently chosen from hydrogen, C₁-C₁₀ alkyl    optionally substituted with hydroxyl and carboxylic acid, C₃-C₆    polyhydroxylated alkyl, C₅-C₁₀ aryl, C₅-C₁₀ heteroaryl, C₃-C₅    heterocycloalkyl optionally substituted with C(O), —(CH₂)_(a)CO₂H    optionally substituted with C₅-C₁₀ heteroaryl, —(CH₂)_(a)CONR³⁰R³¹,    —(CH₂)_(a)NHSO₃ ⁻, —(CH₂)_(a)NHSO₃H, —(CH₂)_(a)OH, —(CH₂)_(a)OPO₃ ⁼,    —(CH₂)_(a)OPO₃H₂, —(CH₂)_(a)OPO₃H⁻, —(CH₂)_(a)OR²², —(CH₂)_(a)OSO₃    ⁻, —(CH₂)_(a)OSO₃H, —(CH₂)_(a)PO₃ ³² , —(CH₂)_(a)PO₃H₂,    —(CH₂)_(a)PO₃H⁻, —(CH₂)_(a)SO₃ ⁻, —(CH₂)_(a)SO₃H,    —(CH₂)_(d)CO(CH₂CH₂O)_(c)R²³, —(CH₂)_(d)(CH₂CH₂O)_(c)R²⁴,    —(CHCO₂H)_(a)CO₂H, —CH₂(CHNH₂)_(a)CH₂NR²⁵R²⁶, —CH₂(CHOH)_(a)CO₂H,    —CH₂(CHOH)_(a)R²⁷, —CH[(CH₂)_(b)NH₂]_(a)CH₂OH,    —CH[(CH₂)_(b)NH₂]_(a)CO₂H, and —(CH₂ _(a)NR²⁸R²⁹;-   each R²² to R³¹ are independently chosen from hydrogen, C₁-C₁₀    alkyl, and C₁-C₅-dicarboxylic acid;-   R³⁵ is chosen from C₁-C₁₀ alkyl optionally substituted with hydroxyl    and carboxylic acid, C₃-C₆ polyhydroxylated alkyl, C₅-C₁₀ aryl,    C₅-C₁₀ heteroaryl, C₃-C₅ heterocycloalkyl optionally substituted    with C(O), —(CH₂)_(a)CO₂H optionally substituted with C₅-C₁₀    heteroaryl, —(CH₂)_(a)CONR³⁰R³¹, —(CH₂)_(a)NHSO₃ ⁻,    —(CH₂)_(a)NHSO₃H, —(CH₂)_(a)OH, —(CH₂)_(a)OPO₃ ⁼, —(CH₂)_(a)OPO₃H₂,    —(CH₂)_(a)OPO₃H⁻, —(CH₂)_(a)OR²², —(CH₂)_(a)OSO₃ ⁻, —(CH₂)_(a)OSO₃H,    —(CH₂)_(a)PO₃ ⁼, —(CH₂)_(a)PO₃H₂, —(CH₂)_(a)PO₃H⁻, —(CH₂)_(a)SO₃ ⁻,    —(CH₂)_(a)SO₃H, —(CH₂)_(d)CO(CH₂CH₂O)_(c)R²³,    —(CH₂)_(d)(CH₂CH₂O)_(c)R²⁴, —(CHCO₂H)_(a)CO₂H,    —CH₂(CHNH₂)_(a)CH₂NR²⁵R²⁶, —CH₂(CHOH)_(a)CO₂H, —CH₂(CHOH)_(a)R²⁷,    —CH[(CH₂)_(b)NH₂]_(a)CH₂OH, —CH[(CH₂ _(b)NH₂]_(a)CO₂H, and    —(CH₂)_(a)NR²⁸R²⁹;-   (AA) is a polypeptide chain comprising one or more natural or    unnatural amino acids linked together by peptide bonds;-   (PS) is a sulfated or non-sulfated polysaccharide chain comprising    one or more monosaccharide units connected by glycosidic linkages;    and-   each ‘a’, ‘b’, and ‘d’ are independently chosen from 0 to 10, ‘c’ is    chosen from 1 to 100 and each of ‘m’ and ‘n’ independently is an    integer from 1 to 3;    irradiating the subject's vasculature with non-ionizing radiation,    wherein the radiation causes the compound to fluoresce;    detecting the fluorescence of the compound in the subject's    vasculature; and    assessing the location of disease or injury in the subject's    vasculature, based on the detected fluorescence.

In an embodiment, said amino acids are chosen from α-amino acids,β-amino acids, and γ-amino acids.

In certain embodiments, the composition is administered intravenously.

In certain embodiments, the non-ionizing radiation has a wavelength ofat least 350 nm.

In certain embodiments, the detected fluorescence of the compound in thesubject's vasculature is measured over time.

In certain embodiments, wherein the subject's pulmonary and cardiacvasculature is assessed. In some embodiments, assessing the subject'spulmonary and cardiac vasculature comprises identifying abnormalitieschosen from stenosis, occlusions, aneurysms, and combinations thereof.In some embodiments, assessing the subject's pulmonary and cardiacvasculature comprises comparing the detected fluorescence in thesubject's pulmonary and cardiac vasculature to that of normal pulmonaryand cardiac vasculature under similar conditions. In some embodiments,wherein the subject's eye vasculature is assessed. In particularembodiments, assessing the subject's eye vasculature comprisesidentifying ocular abnormalities. In particular embodiments, the ocularabnormalities are chosen from blood vessel architecture, ischemic spots,choroidal infarcts, Elschnig's spots, exudates, hemorrhages, andcombinations thereof. In particular embodiments, the ocularabnormalities are chosen from vessel crossovers, vessel tortuosity,evidence of exudates, and combinations thereof. In particularembodiments, assessing the subject's eye vasculature comprises comparingthe detected fluorescence in the subject's eye to that of a normal eyeunder similar conditions.

In accordance with the present invention, one protocol for assessingphysiological function of body cells includes administering an effectiveamount of a pyrazine derivative represented by Formula 1 into a body ofa patient. An appropriate dosage of the pyrazine derivate that isadministered to the patient is readily determinable by one of ordinaryskill in the art and may vary according to the clinical procedurecontemplated, generally ranging from about 1 nanomolar to about 100micromolar. The administration of the pyrazine derivative to the patientmay occur in any of a number of appropriate fashions including, but notlimited to: (1) intravenous, intraperitoneal, or subcutaneous injectionor infusion; (2) oral administration; (3) transdermal absorption throughthe skin; and (4) inhalation.

Still referring to the above-mentioned protocol, the pyrazine derivativeis exposed to visible and/or near infrared light. This exposure of thepyrazine derivate to light may occur at any appropriate time butpreferably occurs while the pyrazine derivative is in the body (e.g., inthe bloodstream) of a patient. Due to this exposure of the pyrazinederivate to the visible and/or infrared light, the pyrazine derivateemanates spectral energy (e.g., visible and/or near infrared light) thatmay be detected by appropriate detection equipment. The spectral energyemanated from the pyrazine derivative tends to exhibit a wavelengthrange greater than a wavelength range absorbed by the pyrazinederivative. For example, if an embodiment of the composition absorbslight of about 700 nm, the composition may emit light of about 745 nm.

Detection of the pyrazine derivate (or more particularly, the lightemanating therefrom) may be achieved through optical fluorescence,absorbance or light scattering procedures known in the art. In oneembodiment, this detection of the emanated spectral energy may becharacterized as a collection of the emanated spectral energy and ageneration of electrical signal indicative of the collected spectralenergy. The mechanism(s) utilized to detect the spectral energy from thecomposition that is present in the body may be designed to detect onlyselected wavelengths (or wavelength ranges) and/or may include one ormore appropriate spectral filters. Various catheters, endoscopes, earclips, hand bands, head bands, surface coils, finger probes and the likemay be utilized to expose the pyrazine derivatives to light and/or todetect the light emanating therefrom. This detection of spectral energymay be accomplished at one or more times intermittently or may besubstantially continuous.

Renal function of the patient can be determined based on the detectedspectral energy using methods described in U.S. Pat. No. 9,216,963,which is hereby incorporated by reference. This can be achieved by usingdata indicative of the detected spectral energy and generating anintensity/time profile indicative of a clearance of the pyrazinederivative from the body. This profile may be correlated to aphysiological or pathological condition. For example, the patient'sclearance profiles and/or clearance rates may be compared to knownclearance profiles and/or rates to assess the patient's renal functionand to diagnose the patient's physiological condition. In the case ofanalyzing the presence of the pyrazine derivative in bodily fluids,concentration/time curves may be generated and analyzed (preferably inreal time) using an appropriate microprocessor to diagnose renalfunction.

Physiological function can be assessed by: (1) comparing differences inmanners in which normal and impaired cells remove a composition of theinvention from the bloodstream; (2) measuring a rate or an accumulationof a composition of the invention in the organs or tissues; and/or (3)obtaining tomographic images of organs or tissues having a compositionof the invention associated therewith. For example, blood pool clearancemay be measured non-invasively from convenient surface capillaries suchas those found in an ear lobe or a finger or can be measured invasivelyusing an appropriate instrument such as an endovascular catheter.Accumulation of a composition of the invention within cells of interestcan be assessed in a similar fashion.

A modified pulmonary artery catheter may also be utilized to, interalia, make the desired measurements of spectral energy emanating from acomposition of the invention. The ability for a pulmonary catheter todetect spectral energy emanating from a composition of the invention isa distinct improvement over current pulmonary artery catheters thatmeasure only intravascular pressures, cardiac output and other derivedmeasures of blood flow. Traditionally, critically ill patients have beenmanaged using only the above-listed parameters, and their treatment hastended to be dependent upon intermittent blood sampling and testing forassessment of renal function. These traditional parameters provide fordiscontinuous data and are frequently misleading in many patientpopulations.

Modification of a standard pulmonary artery catheter only requiresmaking a fiber optic sensor thereof wavelength-specific. Catheters thatincorporate fiber optic technology for measuring mixed venous oxygensaturation exist currently. In one characterization, it may be said thatthe modified pulmonary artery catheter incorporates awavelength-specific optical sensor into a tip of a standard pulmonaryartery catheter. This wavelength-specific optical sensor can be utilizedto monitor renal function-specific elimination of a designed opticallydetectable chemical entity such as the compositions of the presentinvention. Thus, by a method analogous to a dye dilution curve,real-time renal function can be monitored by the disappearance/clearanceof an optically detected compound.

Disclosed herein is a method of optically diagnosing renal function in apatient, comprising the steps of:

-   -   administering a pyrazine derivative or a        pharmaceutically-acceptable salt thereof to a patient;    -   exposing the administered pyrazine derivative to visible and/or        near infrared light;    -   detecting spectral energy emanating from the administered        pyrazine derivative; and    -   determining clearance of the pyrazine derivative from the body        of the patient;    -   wherein the administered pyrazine derivative absorbs and        emanates spectral energy in the visible and/or near infrared        spectrum.

Among some of the various aspects of the present invention is the use ofone or more optical dyes in a surgical procedure to enable a surgeon orother health care professional to demarcate a tissue of the renal systemusing methods described in U.S. Pat. No. 9,283,288, which is herebyincorporated by reference. Advantageously, the surgeon or other healthcare professional can thereby avoid, target and/or assess the integrityof the tissue before, during and/or after the surgical procedure.

One aspect of the present invention is directed to a process for usingan optical agent in a surgical procedure. In this process, a renallyexcretable optical agent is administered to a patient to cause theoptical agent to appear in the patient's urine. Further, a first tissueof the patient's renal system is irradiated with non-ionizing radiation,and the agent is optically detected in the irradiated first tissue todemarcate the position of the first tissue (e.g., relative tosurrounding and/or adjacent tissue).

Another aspect of the invention is directed a process for using anoptical agent in a surgical procedure. In this process, a surgical fieldof a patient is irradiated with non-ionizing radiation while a renallyexcretable optical agent is located in a first tissue of the patient'srenal system in the surgical field. The first tissue is irradiated todetect the optical agent in the first tissue. A second tissue of thepatient is then surgically manipulated based, at least in part, on theoptical detection of the agent in the first tissue.

Yet another aspect of the invention is directed to a process for usingan optical agent in a surgical procedure. In this process, a renallyexcretable optical agent is delivered to at least one tissue of a renalsystem of a patient, and the tissue(s) is(are) irradiated withnon-ionizing radiation. The optical agent is detected (based, at leastin part, on irradiation of the tissue) to determine if the agent isretained within the tissue(s) of the renal system of the patient.

Disclosed herein is a process for using a fluorescent agent in anabdominal or pelvic surgical procedure to reduce the risk of accidentalinjury to a ureter of a patient undergoing an abdominal or pelvicsurgical procedure, the process comprising:

-   -   delivering a fluorescent agent to the ureter of a patient        undergoing an abdominal or pelvic surgical procedure,    -   wherein the fluorescent agent is delivered intravenously,        enterally, intraperitoneally, transdermally, or via inhalation        to the patient,    -   wherein at least a fraction of the fluorescent agent is excreted        through the renal system such that at least a fraction of the        fluorescent agent is present in the patient's urine,    -   wherein the fluorescent agent is chosen from phenylxanthenes,        phenothiazines, phenoselenazines, cyanines, indocyanines,        squaraines, dipyrrolo pyrimidones, anthraquinones, tetracenes,        quinolines, pyrazines, acridines, acridones, phenanthridines,        azo dyes, rhodamines, phenoxazines, azulenes, azaazulenes,        triphenyl methane dyes, indoles, benzoindoles,        indocarbocyanines, benzoindocarbocyanines, derivatives having        the general structure of        4,4-difluoro-4-bora-3a,4a-diaza-s-indacene, and conjugates        thereof and derivatives thereof,    -   irradiating a ureter of the patient's renal system with        non-ionizing radiation to cause the fluorescent agent to        fluoresce within the ureter; and    -   optically detecting the fluorescent agent in the irradiated        ureter to distinguish the ureter from surrounding tissues and        reduce the risk of accidental injury to the ureter.

Kits

The present disclosure provides a kit for assessing vasculature,diagnosing renal function, or using a fluorescent agent in an abdominalor pelvic surgical procedure to reduce the risk of accidental injury toa ureter of a patient in a subject in need thereof, comprising: acompound of structural Formula I

or a salt thereof; wherein or a salt thereof;

-   -   X¹ and X² are independently chosen from —CO(AA), —CN, —CO₂R¹,        —CONR²R³, —COR⁴, —NO₂, —SOR³⁵, —SO₂R⁶, —SO₂R⁷ and —PO₃R⁸R⁹;    -   Y¹ and Y² are independently chosen from —OR¹⁰, —SR¹¹, —NR¹²R¹³,        —N(R¹⁴)COR¹⁵, —CONH(PS); —P(R¹⁶)₂, —P(OR¹⁷)₂ and

-   -   each Z¹ is independently chosen from a bond, —CR¹⁸R¹⁹, —O—,        —NR²⁰—, —NCOR²¹—, —S—, —SO—, and —SO₂—;    -   each R¹ to R²¹ are independently chosen from hydrogen, C₁-C₁₀        alkyl optionally substituted with hydroxyl and carboxylic acid,        C₃-C₆ polyhydroxylated alkyl, C₅-C₁₀ aryl, C₅-C₁₀ heteroaryl,        C₃-C₅ heterocycloalkyl optionally substituted with C(O),        —(CH₂)_(a)CO₂H optionally substituted with C₅-C₁₀ heteroaryl,        —(CH₂)_(a)CONR³⁰R³¹, —(CH₂)_(a)NHSO₃ ⁻, —(CH₂)_(a)NHSO₃H,        —(CH₂)_(a)OH, —(CH₂)_(a)OPO₃ ⁼, —(CH₂)_(a)OPO₃H₂,        —(CH₂)_(a)OPO₃H^(−l , —(CH) ₂)_(a)OR²², —(CH₂)_(a)OSO₃ ⁻,        —(CH₂)_(a)OSO₃H, —(CH₂)_(a)PO₃ ⁼, —(CH₂)_(a)PO₃H₂,        —(CH₂)_(a)PO₃H⁻, —(CH₂)_(a)SO₃ ⁻, —(CH₂)_(a)SO₃H,        —(CH₂)_(d)CO(CH₂CH₂O)_(c)R²³, —(CH₂)_(d)(CH₂CH₂O)_(c)R²⁴,        —(CHCO₂H)_(a)CO₂H, —CH₂(CHNH₂)_(a)CH₂NR²⁵R²⁶,        —CH₂(CHOH)_(a)CO₂H, —CH₂(CHOH)_(a)R²⁷, —CH        [(CH₂)_(b)NH₂]_(a)CH₂OH, —CH[(CH₂)_(b)NH₂]_(a)CO₂H, and        —(CH₂)_(a)NR²⁸R²⁹;    -   each R²² to R³¹ are independently chosen from hydrogen, C₁-C₁₀        alkyl, and C₁-C₅-dicarboxylic acid;    -   R³⁵ is chosen from C₁-C₁₀ alkyl optionally substituted with        hydroxyl and carboxylic acid, C₃-C₆ polyhydroxylated alkyl,        C₅-C₁₀ aryl, C₅-C₁₀ heteroaryl, C₃-C₅ heterocycloalkyl        optionally substituted with C(O), —(CH₂)_(a)CO₂H optionally        substituted with C₅-C₁₀ heteroaryl, —(CH₂)_(a)CONR³⁰R³¹,        —(CH₂)_(a)NHSO₃ ⁻, —(CH₂)_(a)NHSO₃H, —(CH₂)_(a)OH,        —(CH₂)_(a)OPO₃ ⁼, —(CH₂)_(a)OPO₃H₂, —(CH₂)_(a)OPO₃H⁻,        —(CH₂)_(a)OR²², —(CH₂)_(a)OSO₃ ⁻, —(CH₂)_(a)OSO₃H, —(CH₂)_(a)PO₃        ⁼, —(CH₂)_(a)PO₃H₂, —(CH₂)_(a)PO₃H⁻, —(CH₂)_(a)SO₃ ⁻,        —(CH₂)_(a)SO₃H, —(CH₂)_(d)CO(CH₂CH₂O)_(c)R²³,        —(CH₂)_(d)(CH₂CH₂O)_(c)R²⁴, —(CHCO₂H)_(a)CO₂H,        —CH₂(CHNH₂)_(a)CH₂NR²⁵R²⁶, —CH₂(CHOH)_(a)CO₂H,        —CH₂(CHOH)_(a)R²⁷, —CH[(CH₂)_(b)NH₂]_(a)CH₂OH,        —CH[(CH₂)_(b)NH₂]_(a)CO₂H, and —(CH₂)_(a)NR²⁸R²⁹,    -   (AA) is a polypeptide chain comprising one or more natural or        unnatural amino acids linked together by peptide bonds;    -   (PS) is a sulfated or non-sulfated polysaccharide chain        comprising one or more monosaccharide units connected by        glycosidic linkages; and    -   each ‘a’, ‘b’, and ‘d’ are independently chosen from 0 to 10,        ‘c’ is chosen from 1 to 100 and each of ‘m’ and ‘n’        independently is an integer from 1 to 3; and        written instructions for assessing the vasculature in the        subject, comprising the steps of:    -   administering an effective amount of the compound of structural        Formula I;    -   irradiating the subject's vasculature with non-ionizing        radiation, wherein the radiation causes the compound to        fluoresce;    -   detecting the fluorescence of the compound in the subject's        vasculature; and    -   visualizing the vasculature within the subject based on the        detected fluorescence.

In an embodiment, said amino acids are chosen from α-amino acids,β-amino acids, and γ-amino acids.

In certain embodiments, the instructions include a step foradministering the compound intravenously.

In certain embodiments, the instructions include a step for irradiatingthe composition with the non-ionizing radiation having a wavelength ofat least 350 nm.

In certain embodiments, the instructions include a step for measuringthe detected fluorescence of the compound in the subject's vasculatureover time.

In certain embodiments, the subject's pulmonary and cardiac vasculatureis assessed.

In certain embodiments, the instructions include a step for assessingthe subject's pulmonary and cardiac vasculature comprising identifyingabnormalities chosen from stenosis, occlusions, aneurysms, andcombinations thereof

In certain embodiments, the instructions include a step for assessingthe subject's pulmonary and cardiac vasculature comprising comparing thedetected fluorescence in the subject's pulmonary and cardiac vasculatureto that of normal pulmonary and cardiac vasculature under similarconditions.

In certain embodiments, the instructions include a step for thesubject's eye vasculature to be assessed.

In certain embodiments, the instructions include a step for assessingthe subject's eye vasculature comprises identifying ocularcharacteristics. In particular embodiments, the ocular characteristicsare chosen from blood vessel architecture, ischemic spots, choroidalinfarcts, Elschnig's spots, exudates, hemorrhages, and combinationsthereof. In particular embodiments, the ocular characteristics arechosen from vessel crossovers, vessel tortuosity, evidence of exudates,and combinations thereof.

In certain embodiments, the instructions include a step for assessingthe subject's eye vasculature comprises comparing the detectedfluorescence in the subject's eye to that of a normal eye under similarconditions.

Formulation

The compositions of the present disclosure may be administeredintravenously, intraperitoneally, or via subcutaneous injection orinfusion; so that the compound enters the bloodstream.

Pyrazine derivatives of this invention can be administered as solutionsin most pharmaceutically acceptable intravenous vehicles known in theart. Pharmaceutically acceptable vehicles that are well known to thoseskilled in the art include, but are not limited to, 0.01-0.1 M phosphatebuffer or 0.8% saline. Additionally, pharmaceutically acceptablecarriers may be aqueous or non-aqueous solutions, suspensions,emulsions, or appropriate combinations thereof. Examples of non-aqueoussolvents are propylene glycol, polyethylene glycol, vegetable oils suchas olive oil, and injectable organic esters such as ethyl oleate.Examples of aqueous carriers are water, alcoholic/aqueous solutions,emulsions or suspensions, including saline and buffered media. Exemplaryparenteral vehicles include sodium chloride solution, Ringer's dextrose,dextrose and sodium chloride, lactated Ringer's or fixed oils. Exemplaryintravenous vehicles include fluid and nutrient replenishers,electrolyte replenishers such as those based on Ringer's dextrose, andthe like. Preservatives and other additives may also be present, suchas, for example, antimicrobials, antioxidants, collating agents, inertgases and the like.

Dosage

Compositions of the present disclosure may be administered in a singledose or in multiple doses to achieve an effective diagnostic objective.After administration, the composition is allowed time to move into theeye, and the selected target site is exposed to light with a sufficientpower and intensity to detect light emanating from the compound withinthe patient's body to provide information that may be utilized by ahealthcare provider (e.g., in making a diagnosis). Doses may vary widelydepending upon, for example, the particular integrated photoactive agentemployed, the areas (e.g., organs or tissues) to be examined, theequipment employed in the clinical procedure, the efficacy of thetreatment achieved, and/or the like. For example, the dosage of thecompound may vary from about 0.1 mg/kg body weight to about 500 mg/kgbody weight in some embodiments. In other embodiments, the dosage of thecompound may vary from about 0.5 to about 2 mg/kg body weight.

Detection and Measurement

For detection of vasculature, after administration, the composition isallowed time to move into the eye, and the selected target site isexposed to light with a sufficient power and intensity to detect lightemanating from the compound within the patient's body to provideinformation that may be utilized by a healthcare provider (e.g., inmaking a diagnosis). Detection of the composition may be achieved byoptical fluorescence, absorbance, and/or light scattering methods. Thisdetection of spectral energy may be accomplished at one or more timesintermittently or may be substantially continuous.

In an embodiment, the process of the present invention can be used toenable the surgeon or other healthcare individual to avoid theureter(s), the bladder, and/or the urethra. In a healthy individual,urine flows from the kidneys through the ureter and collects in thebladder, where it is stored until it is eliminated from the body throughthe urethra. Thus, in the process of the present invention, detection ofthe optical agent(s) in the ureter and bladder is possible due to theaccumulation of the agent(s) in urine present in those structures.Detection of the optical agent(s) in the urethra is possible, forexample, where residue of urine containing the optical agents is presenton the walls of the urethra, or where a urinary catheter facilitatescontinuous flow of urine through the urethra.

Alternatively, another aspect of the present invention is the use of oneor more optical agents to demarcate the target of a surgical procedure.Such surgical procedures include, but are not limited to, for example,nephrectomy, renal transplantation surgery, resection of a ureteralsegment during removal of a tumor, bladder neck suspension surgery, andsurgical removal of kidney stones.

A further aspect of the present invention is the use of the opticalagent(s) to assess the integrity of the renal system. Such an assessmentcan be made before, during, and/or after a surgical procedure performedon the renal system and/or other organ and/or tissue in the abdominaland/or pelvic region. Confinement of the optical agent to the tissues ofthe renal system indicates that no damage to the renal system (e.g.,nicking of the ureter) has occurred. If damage or injury to a tissue ofthe renal system has occurred, the process of the present inventionallows a surgeon to rapidly identify the location of such damage orinjury (e.g., by observing the egress of dye from the site of damage).

A further aspect of the present invention is the use of the opticalagent(s) to detect one or more tissues of the renal system during adiagnostic laparoscopic procedure.

Depending upon the surgical technique employed, the presence of theoptical agent in a first tissue may be detected by irradiating theentire surgical field. This approach could be used, for example, in opensurgical procedures. Alternatively, only a portion of the surgical fieldor the specific site(s) to be monitored may be illuminated, for example,using a laparoscope or other endoscopic tool.

In general, any source of irradiation capable of providing non-ionizingradiation of a desired wavelength may be used. For example, in oneembodiment, the operating room lighting (e.g., fluorescent orincandescent lighting) emits light of the desired wavelength. In anotherembodiment, the source of irradiation is a laser. In yet anotherembodiment, the source of irradiation is a hand-held light. Othersources of irradiation that can be used include, but are not limited to,lighted catheters, endoscopes, fiber optic probes, light emitting diodes(LEDs), lighted headbands (also called headlights), and the like. Asurgical instrument that contains or is configured with an illuminationsystem may also be employed. Examples of such instruments include thefiber optic instruments available from BioSpec (Moscow, Russia) and theTC-I fiber optic tool for photodynamic therapy with fine needle tip forirradiating interstitial tumors (http://www.biospec.ru/_FiberOptics_e.html).

Any of the optical detection methods available in the art can be used inthe present invention. Spectroscopic measurements can be separated intothree broad categories: absorbance, scattering/reflectance, andemission. Absorbance assays involve relating the amount of incidentlight that is absorbed by a sample to the type and number of moleculesin the sample. For example, in case of absorbance measurement, it isdesirable that the wavelength of the non-ionizing radiation that is usedis one which is absorbed by the optical agent. Most commonly, absorbanceis measured indirectly by studying the portion of incident light thatemerges from the sample. Scattering assays are similar to absorbance inthat the measurement is based on the amount of incident light thatemerges or is transmitted from the sample or tissue. However, in thecase of scattering, the signal increases with the number ofinteractions, whereas, in the case of absorbance, the signal isinversely proportional to the interactions. Emission assays look atelectromagnetic emissions from a sample other than the incident light.In each case, the measurements may be broad spectrum orfrequency-specific depending on the particular assay. Most commonly,emission assays involve the measurement of luminescence.

Luminescence is the emission of light from excited electronic states ofatoms or molecules. Luminescence generally refers to all kinds of lightemission, except incandescence, and may include photoluminescence,chemiluminescence, and electrochemiluminescence, among others. Inphotoluminescence, including fluorescence and phosphorescence, theexcited electronic state is created by the absorption of electromagneticradiation. Luminescence assays involve detection and interpretation ofone or more properties of the luminescence or associated luminescenceprocess. These properties include intensity, excitation and/or emissionspectrum, polarization, lifetime, and energy transfer, among others.These properties also include time-independent (steady-state) and/ortime-dependent (time-resolved) properties of the luminescence.Representative luminescence assays include fluorescence intensity(FLINT), fluorescence polarization (FP), fluorescence resonance energytransfer (FRET), fluorescence lifetime (FLT), total internal reflectionfluorescence (TIRF), fluorescence correlation spectroscopy (FCS),fluorescence recovery after photobleaching (FRAP), and bioluminescenceresonance energy transfer (BRET), among others. By way of example, whena fluorescent optical agent is used in the present invention, it isdesirable that the wavelength of non-ionizing radiation be such that itexcites the optical agent. This excitation causes the molecule to emitpart of the absorbed energy at a different wavelength, and the emissioncan be detected using fluorometric techniques as described above. Oneskilled in the art can readily determine the most appropriate detectiontechnique based on, in part, the specific optical agent(s) administered,the tissue to be detected, and the type of surgical procedure involved.For example, in some embodiments, the surgeon will be able to see theoptical agent in the surgical field. Other embodiments employ an opticalagent that can be detected using a laparoscopic instrument.

Upon irradiation with electromagnetic radiation of the properwavelength, an optical agent may be detected by visual or other opticalmeans. For example, optical detection may be achieved using the unaidedeye or by one or more imaging or detecting devices (e.g., a camera,charged coupled device (CCD), photomultiplier tube (PMT), avalanchediode, photodiodes), or detection involving an electronic processingstep (e.g., detecting, enhancing, processing, analyzing, quantitating,or otherwise manipulating a signal using software or other means).

In order that the disclosure described herein may be more fullyunderstood, the following examples are set forth. It should beunderstood that these examples are for illustrative purposes only andare not to be construed as limiting this disclosure in any manner.

EXAMPLES

Non-limiting examples of methods utilizing pyrazinc dyes include thefollowing compounds and pharmaceutically acceptable salts thereof:

Example 1: Assessing Eye Vasculature

An exemplary procedure for assessing eye vasculature is as follows: acomposition containing a fluorescent pyrazinc molecule is administeredinto a subject's bloodstream. While in the blood stream, the fluorescentdye molecule may be irradiated with non-ionizing radiation, wherein theradiation causes the composition to fluoresce. The fluorescence of anyabsorbed challenge molecule may be detected in the bloodstream; and theeye vasculature may be assessed based on the detected fluorescence.

Yet another exemplary procedure may be performed as described above, butin addition, the fluorescence of the fluorescent pyrazine molecule maybe detected in the eye over time; and the location of disease or injuryin the subject's eye may be determined based on the time between thedetected fluorescence of each fluorescent challenge molecule andadministration.

Example 2: 3,6-diamino-N²,N⁵-bis(D-serine)-pyrazine-2,5-dicarboxamide

This fluorescent molecule exhibits light absorption and emission maximaat 445 nm and 560 nm, respectively; and is an example of a fluorescentpyrazine molecule that may used to assess and/or visualize thevasculature of a subject.

Step 1. Synthesis of 3,6diamino-N²,N⁵-bis(O-benzyl-(D)-serine methylester)-pyrazine-2, 5-dicarboxamide

A mixture of sodium 3,6-diaminopyrazine-2,5-dicarboxylate (300 mg, 1.24mmol), (D)-Ser(OBn)-OMe-HCl salt (647 mg, 2.64 mmol), HOBt-H₂O (570 mg,3.72 mmol) and EDC-HCl (690 mg, 3.60 mmol) in DMF (25 mL) was treatedwith TEA (2 mL). The resulting mixture was stirred for 16 h andconcentrated. The mixture was concentrated to dryness and the residuewas partitioned with EtOAc and water. The layers were separated and theEtOAc solution was washed with saturated NaHCO₃ and brine. The solutionwas dried over anhydrous Na₂SO₄, filtered and concentrated, whichafforded 370 mg (51% yield) of the bisamide as a bright yellow powder:¹NMR (300 MHz, CDCl₃) δ 8.47 (d, J=8.74 Hz, 2 H), 7.25-7.37 (complex m,10H), 5.98 (bs, 4 H), 4.85 (dt, J=8.7, 3.3 Hz, 2 H), 4.56 (ABq, J=12.6,Hz, Av=11.9 Hz, 4 H), 3.99 (one half of an ABq of d, J=8.7, 3.3, Avobscured, 2 H), 3.76-3.80 (one half of an ABq-obscured, 2 H), 3.78 (s, 6H). ¹³C NMR (75 MHz, CDCl₃) δ 170.5 (s), 165.1 (s), 146.8 (s), 138.7 (s)128.6 (d), 128.1 (d), 127.8 (d), 126.9 (s), 73.5 (t), 69.8 (t), 53.0(q), 52.9 (q). LCMS (5-95% gradient acetonitrile with 0.1% TFA over 10min), single peak retention time=4.93 min on 30 mm column, (M+H)⁺=581.

Step 2. Synthesis of3,6diamino-N²,N⁵-bis(O-benzyl-(D)-serine)-pyrazine-2,5-dicarboxamide

The product from Step 1 (370 mg, 0.64 mmol) in THF (10 mL) was treatedwith 1.0 N sodium hydroxide (2.5 mL). After stirring at room temperaturefor 30 min, the reaction was judged complete by TLC. The pH was adjustedto approximately 2 by the addition of 1.0 N HCl and the resultingsolution was extracted (3x) with EtOAc. The layers were combined, driedover sodium sulfate, filtered and concentrated to afford 353 mg (100%yield) of the di-acid as an orange foam: LCMS (5-95% gradientacetonitrile in 0.1% TFA over 10 min), retention time=4.41 min on 30 mmcolumn, (M+H)⁺=553.

Step 3. Synthesis of3,6-diamino-N²,N⁵-bis(D-serine)-pyrazine-2,5-dicarboxamide

To the product from Step 2 (353 mg, 0.64 mmol) in methanol (20 mL) wasadded 5% Pd/C (300 mg) and ammonium formate (600 mg). The resultingreaction was heated at reflux for 2 h. The reaction was cooled to roomtemperature, filtered through a plug of celite and concentrated. Theresidue was recrystallized from methanol-ether to provide 191 mg (80%yield) of title compound Example 2 as a yellow foam: ¹NMR (300 MHz,DMSO-d₆) 5 8.48 (d, J=6.9 Hz, 2 H), 6.72 (bs, 4 H), 3.95 (apparentquartet, J=5.1 Hz, 2 H), 3.60 (apparent ABq of doublets; down-fieldgroup centered at 3.71, J=9.9, 5.1 Hz, 2H; up-field group centered at3.48, J=20 9.9, 6.3 Hz, 2 H). ¹³C NMR (75 MHz, CDCl₃) 5 172.9 (s), 164.9(s), 147.0 (s), 127.0 (s), 62.9 (d), 55.7 (t). LCMS (5-95% gradientacetonitrile in 0.1% TFA over 10 min), single peak retention time=1.45min on 30 mm column, (M+H)⁺=373. UV/vis (100 μM in PBS) λ_(abs)=434 nm.Fluorescence λ_(ex)=449 nm, λ_(em)=559 nm.

Example 3: 3,6-diamino-N²,N⁵-bis(L-serine)-pyrazine-2,5-dicarboxamide

The title compound is prepared using the procedures of Example 2 andsubstituting (L)-Ser(OBn)-OMe-HCl salt for (D)-Ser(OBn)-OMe-HCl salt inStep 1. RP-LC/MS (ESI) m/z 373.2 (M+H)⁺. Anal. Calcd for C₁₂H₁₆N₆O₈: C,38.71; H, 4.33; N, 22.57. Found: C, 38.44; H, 4.51; N, 22.33.

This fluorescent molecule exhibits light absorption and emission maximaat 445 nm and 560 nm, respectively.

Example 4: 3,6-diamino-N²,N²,N⁵,N⁵-tetrakis(2-methoxtethyl)pyrazine-2,5-dicarboxamide

A mixture of 3,6-diaminopyrazine-2,5-dicarboxylic acid (200 mg, 1.01mmol), bis-2-(methoxyethyl) amine (372 mL, 335.5 mg, 2.52 mmol),HOBt-H₂O (459 mg, 3.00 mmol), and EDC-HCl (575 mg, 3.00 mmol) werestirred together in DMF (20 mL) for 1 h at room temperature. The mixturewas concentrated to dryness and the residue was partitioned with EtOAcand water. The layers were separated and the EtOAc solution was washedwith saturated NaHCO₃ and brine. The solution was dried over anhydrousNa₂SO₄, filtered and concentrated. Purification by radial flashchromatography (SiO₂, 10/1 CHCl₃-MeOH) afforded 228.7 mg (53% yield) ofExample 4 as an orange foam: ¹H NMR (300 MHz, CDCl₃), δ 4.92 (s, 4 H),3.76 (apparent t, J=5.4 Hz, 4 H), 3.70 (apparent t, J=5.6 Hz, 4 H), 3.64(apparent t, J=5.4 Hz, 4 H), 3.565 (apparent t, J=5.4 Hz), 3.67 (s, 6H), 3.28 (s, 6 H). ¹³C NMR (75 MHz, CDCL3) □ 167.6 (s), 145.6 (s), 131.0(s), 72.0 (t), 70.8 (t), 59.2 (q), 49.7 (t), 47.1 (t). LCMS (5-95%gradient acetonitrile in 0.1% TFA over 10 min), single peak retentiontime=3.14 min on 30 mm column, (M+H)⁺=429. UV/vis (100 μM in PBS)λ_(abs)=394 nm. Fluorescence (100 μm) λ_(ex)=394 nm λ_(em)=550 nm.

Example 5: 3,6-diamino-N²,N⁵-bis (2,3-dihydroxypropyl)pyrazine-2,5-dicarboxamide

Step 1. Synthesis of3,6-diamino-N²,N⁵-bis((2,2-dimethyl-1,3-dioxolan-4-yl)methyl)pyrazine-2,5-dicarboxamide.

A mixture of 3,6-diaminopyrazine-2,5-dicarboxylic acid (350 mg, 1.77mmol), racemic (2,2-dimethyl-1,3-dioxolan-4-yl)methanamine (933 μL, 944mg, 7.20 mmol), HOBt-H₂O (812 mg, 5.3 mmol), and EDC-HCl (1.02 g, 5.32mmol) were stirred together in DMF (20 mL) for 16 h at room temperature.The mixture was concentrated to dryness and the residue was partitionedwith EtOAc and water. The layers were separated and the EtOAc solutionwas washed with saturated NaHCO₃ and brine. The solution was dried overanhydrous Na₂SO₄, filtered and concentrated to afford 665 mg (88% yield)of the bis-amide diastereomeric pair as a yellow solid: ¹NMR (300 MHz,CDCl₃) δ 8.38 (t, J=5.8 Hz, 2 H), 6.55 (s, 4 H), 4.21 (quintet, J=5.8Hz, 2 H), 3.98 (dd, J=8.4 Hz, 6.3 Hz, 2 H), 3.65 (dd, J=8.4 Hz, J=5.8Hz, 2 H), 3.39 (apparent quartet-diastereotopic mixture, J=5.9 Hz, 4 H),1.35 (s, 6 H), 1.26 (s, 6 H). ¹³C NMR (75 MHz, CDCl₃) δ 165.7 (s), 146.8(s), 126.8 (s), 109.2 (s), 74.8 (d), 67.2 (t), 42.2, 41.1(t-diastereotopic pair), 27.6 (q), 26.1 (q).

Step 2. Synthesis of 3,6-diamino-N²,N⁵-bis (2,3-dihydroxypropyl)pyrazine-2,5-dicarboxamide

The product from Step 1 was dissolved in THF (100 mL) and treated with1.0 N HCl (2 mL). After hydrolysis was complete, the mixture was treatedwith K₂CO₃ (1 g) and stirred for 1 h and filtered through a plug of C18silica using methanol. The filtrate was concentrated to dryness and theresidue was triturated with MeOH (50 mL). The solids were filtered anddiscarded and the residue was treated with ether (50 mL). Theprecipitate was collected by filtration and dried at high vacuum. Thismaterial was purified by radial flash chromatography to afford 221 mg(36% yield) of Example 5 as an orange solid: ¹NMR (300 MHz, DMSO-d₆) δ8.00 (bm, 6 H), 5.39 (bs, 2 H), 4.88 (bs, 2 H), 3.63-3.71 (complex m, 2H), 3.40 (dd, J=11.1, 5.10 Hz, 2 H), 3.28 (dd, J=11.1, 6.60 Hz, 2 H),2.92 (dd, J=12.6, 3.3 Hz, 2 H), 2.65 (dd, J=12.6, 8.4 Hz, 2 H). LCMS(5-95% gradient acetonitrile in 0.1% TFA over 10 min), single peakretention time=4.13 min on 30 mm column, (M+H)⁺=345. UV/vis (100 μM inH₂O) λ_(abs)=432 nm. Fluorescence λ_(ex)=432 nm, λ_(em)=558 nm.

Example 6:3,6-bis(bis(2-methoxyethyl)amino-N²,N²,N⁵,N⁵-tetrakis(2-methoxyethyl)pyrazine-2,5-dicarboxamide bis TFA salt

Step 1. Synthesis of 3,6-dibromopyrazine-2,5-dicarboxylic acid

3,6-Diaminopyrazine-2,5-dicarboxylic acid (499 mg, 2.52 mmol) wasdissolved in 48% hydrobromic acid (10 mL) and cooled to 0° C. in anice-salt bath. To this stirred mixture was added a solution of sodiumnitrite (695 mg, 10.1 mmol) in water (10 mL) dropwise so that thetemperature remains below 5° C. The resulting mixture was stirred for 3h at 5-15° C., during which time the red mixture became a yellowsolution. The yellow solution was poured into a solution of cupricbromide (2.23 g, 10.1 mmol) in water (100 mL) and the resulting mixturewas stirred at room temperature. After an additional 3 h, the aqueousmixture was extracted with EtOAc (3×). The combined extracts were dried(Na₂SO₄), filtered and concentrated to afford 440 mg (54% yield)3,6-dibromopyrazine-2,5-dicarboxylic acid as a pale yellow solid: ¹³CNMR (75 MHz, CDCl₃) δ 164.3 (s), 148.8 (s), 134.9 (s). HPLC (5-95%gradient acetonitrile in 0.1% TFA over 10 min), single peak retentiontime=2.95 min on 250 mm column.

Step 2. Synthesis of3-(Bis(2-methoxyethyl)amino)-6-bromo-N²,N²,N⁵,N⁵-tetrakis(2-methoxyethyl)pyrazine-2,5-dicarboxamide

The product from step 1 (440 mg, 1.36 mmol) was dissolved in DMF (25mL), treated with HOBt-H₂O (624 mg, 4.08 mmol), and EDC-HCl (786 mg,4.10 mmol) and stirred for 30 min at 5 room temperature.Bis(2-methoxyethyl) amine (620 mL, 559 mg, 4.20 mmol) was added and theresulting mixture was stirred at room temperature for 16 h andconcentrated. The residue was partitioned with water and EtOAc. TheEtOAc layer was separated and the aqueous was extracted again withEtOAc. The combined organic layers were washed with 0.5 N HCl, saturatedsodium bicarbonate, and brine. The organic layer was dried (Na/SO₄),filtered and concentrated to afford 214 mg of3-(bis(2-methoxyethypamino)-6-bromo-N2,N2,N5,N5-tetrakis(2-methoxyethyl) pyrazine-2,5-dicarboxamide (26% yield) as a brown oil:LCMS (5¬95% gradient acetonitrile in 0.1% TFA over 10 min), single peakretention time=3.85 min on 30 mm column, (M+H)+=608.

Step 3. Synthesis of3,6-bis(bis(2-methoxyethyl)amino-N²,N²,N⁵,N⁵-tetrakis(2-methoxyethyl)pyrazine-2,5-dicarboxamide bis TFA salt

To the product from step 2 (116 mg, 0.19 mmol) was addedbis(2-methoxylethyl)amine (3.0 mL, 2.71 g, 20.3 mmol) and a “spatulatip” of Pd(PPh₃)₄. The resulting mixture was heated to 140° C. for 2 h.The reaction was cooled and concentrated. The residue was purified byflash chromatography (SiO₂, 10/1 CHCl₃-MeOH). The resulting material waspurified again by reverse phase medium pressure reverse phasechromatography (C18, 10-50% manual gradient acetonitrile in 0.1% TFA) toafford 12 mg (10% yield) of Example 6 as an orange-brown film: LCMS(15-95% gradient acetonitrile in 0.1% TFA over 10 min), single peakretention time=3.85 min on 250 mm column, (M+H)+=661. UV/vis (100 μM inPBS) λ_(abs)=434 nm. Fluorescence λ_(ex)=449 nm, λ_(em)=559 nm.

Example 7: 3,6-diamino-N²,N⁵-bis(2-aminoethyl)pyrazine-2,5-dicarboxamideBis TFA Salt

Step 1. Synthesis of3,6-diamino-N²,N⁵-bis[2-(tert-butoxycarbonyl)aminoethyl]pyrazine-2,5-dicarboxamide

A mixture of sodium 3,6-diaminopyrazine-2,5-dicarboxylate (500 mg, 2.07mmol), tert-butyl 2-aminoethylcarbamate (673 mg, 4.20 mmol), HOBt-H₂O(836 mg, 5.46 mmol) and EDC-HCl (1.05 g, 5.48 mmol) in DMF (25 mL) wasstirred for 16 h and concentrated. Work up as in Example 2 afforded 770mg (76% yield) of the bisamide as an orange foam: ¹NMR (300 MHz,DMSO-d⁶) major comformer, δ 8.44 (t, J=5.7 Hz, 2 H), 6.90 (t, J=5.7 Hz,2 H), 6.48 (bs, 4 H), 2.93-3.16 (complex m, 8 H), 1.37 (s, 9 H), 1.36(s, 9 H). ¹³C NMR (75 MHz, DMSO-d6), conformational isomers δ 165.1 (s),155.5 (bs), 155.4 (bs), 146.0 (s), 126.2 (s), 77.7 (bs), 77.5 (bs), 45.2(bt), 44.5 (bt). 28.2 (q).

Step 2. Synthesis of3,6-diamino-N²,N⁵-bis(2-aminoethyl)pyrazine-2,5-dicarboxamide bis TFAsalt

To the product from step 1 (770 mg, 1.60 mmol) in methylene chloride(100 mL) was added TFA (25 mL) and the reaction was stirred at roomtemperature for 2 h. The mixture was concentrated and the residue takenup into methanol (15 mL). ether (200 mL) was added and the orange solidprecipitate was isolated by filtration and dried at high vacuum toafford 627 mg (77% yield) of Example 7 as an orange powder: ¹NMR (300MHz, DMSO-d₆) δ 8.70 (t, J=6 Hz, 2H), 7.86 (bs, 6H), 6.50 (bs, 4H),3.46-3.58 (m, 4H), 3.26-3.40 (m, 4H). ¹³C NMR (75 MHz, DMSO-d6) δ 166.4(s), 146.8 (s), 127.0 (s), 39.4 (t), 37.4 (t). LCMS (5-95% gradientacetonitrile in 0.1% TFA over 10 min), single peak retention time=3.62min on 30 mm column, (M+H)+=283. UV/vis (100 μM in PBS) λ_(abs)=435 nm.Fluorescence (100 nM) λex=449 nm, λ_(em)=562.

Example 8: 3,6-diamino-N²,N⁵-bis(D-aspartate)-pyrazine-2,5-dicarboxamide

Step 1. Synthesis of 3,6-diamino-N²,N⁵-bis(benzylD-O-benzyl-Aspartate)-pyrazine-2, 5-dicarboxamide

A mixture of sodium 3,6-diaminopyrazine-2,5-dicarboxylate (600 mg, 2.48mmol), Asp(OBn)-OMe-p-TosH salt (2.43 g, 5.00 mmol), HOBt-H₂O (919 mg,6.00 mmol) and EDC-HCl (1.14 g, 5.95 mmol) in DMF (50 mL) was treatedwith TEA (4 mL). The resulting mixture was stirred over night at roomtemperature. The reaction mixture was concentrated and the residue waspartitioned with water and EtOAc. The EtOAc layer was separated andwashed successively with saturated sodium bicarbonate, water and brine.The EtOAc solution was dried (Na₂SO₄), filtered and concentrated. Theresidue was purified by flash chromatography (SiO₂, 50/1 CHCl₃-MeOH to10/1) to afford 1.15 g of the bis-amide (58% yield) as a yellow foam:¹NMR (500 MHz, CDCl₃) 67 8.61 (d, J=8.4 Hz, 2H), 7.29-7.39 (m, 20H),5.85 (bs, 4H), 5.22 (ABq, J=10.0 Hz, Av=17.3 Hz, 4H), 5.10 (ABq, J=12.2Hz, Av=34.3 Hz, 4H), 5.06-5.09 (obs m, 2H), 3.11 (ABq of d, J=17.0, 55.14 Hz, Av=77.9 Hz, 4H). ¹³C NMR (75 MHz, CDCl₃) δ 170.7 (s), 170.7(s), 165.4 (s), 147.0 (s), 135.7 (s), 135.6 (s), 129.0 (d), 128.9 (d),128.8 (d), 128.75 (d), 128.7 (d), 126.9 (s), 68.0 (t), 67.3 (t), 49.1(d), 37.0 (t). LCMS (50-95% 10 gradient acetonitrile in 0.1% TFA over 10min), single peak retention time=5.97 min on 250 mm column, (M+H)+=789.

Step 2. Synthesis of3,6-diamino-N²,N⁵-bis(D-aspartate)-pyrazine-2,5-dicarboxamide

To the product from Step 1 (510mg, 0.65 mmol) was added THF (20 mL) andwater (10 mL). To this stirred mixture was added 10% Pd(C) (500 mg) andammonium formate (1 g). The resulting mixture was heated to 60° C. for 2h and allowed to cool to room temperature. The mixture was filteredthrough celite and concentrated. The resulting material was purifiedagain by reverse phase medium pressure chromatography (C18, 10-70%manual gradient acetonitrile in 0.1% TFA) to afford 137.8 mg (54% yield)of Example 8 as an orange solid: ¹NMR (300 MHz, DMSO-d6) 8 8.62 (d,J=8.4 Hz, 2H), 6.67 (bs, 4H), 4.725 (dt, J=8.4, 5.4 Hz, 2H), 252.74-2.88 (complex m, 4H). ¹³C NMR (75 MHz, DMSO-d6) δ 172.6 (s), 165.2(s), 147.0 (s), 126.6 (s), 60.8 (t), 49.1 (d). LCMS (5-95% gradientacetonitrile in 0.1% TFA over 10 min), single peak retention time=4.01min on 250 mm column, (M+H)+=429. UV/vis (100 μM in PBS) λab=433 nm.Fluorescence (100 nM) λ_(ex)=449 nm, λ_(em)=558 nm.

Example 9:3,6-diamino-N²,N⁵-bis(14-oxo-2,5,8,11-tetraoxa-15-azaheptadecan-17-yl)pyrazine-2,5-dicarboxamide

To a solution of Example 7 (77.4 mg, 0.15 mmol) in DMF 50 (5 mL) wasadded TEA (151 mg, 1.49 mmol) and 2,5-dioxopyrrolidin-1-yl 2,5,8,11-tetraoxatetradecan-14-oate (113 mg, 0.34 mmol) and the reaction wasstirred for 16 h at room temperature. The reaction was concentrated andthe residue was purified by medium pressure reversed phasechromatography (LiChiroprep RP-18 Lobar (B) 25×310 mm—EMD chemicals40-63 μm, ˜70 g, 90/10 to 80/20 0.1% TFA-ACN) to afford 37.4 mg (35%yield) of Example 9 as an orange film: ¹NMR (300 MHz, DMSO-d₆) δ 8.47(t, J=5.7 Hz, 2H), 7.96 (t, J=5.4 Hz, 2H), 3.20-3.60 (complex m, 36 H),3.47 (s, 3H), 60 3.46 (s, 3H), 2.30 (t, J=6.3 Hz, 4H). ¹³C NMR (75 MHz,DMSO-d6) δ 170.2 (s), 165.1 (s), 146.0 (s), 126.2 (s), 71.2 (t), 69.7(t), 69.6 (t), 69.5 (t), 69.4 (t), 66.7 (t), 58.0 (q), 38.2 (t), 36.2(t). LCMS (5-95% gradient acetonitrile in 0.1% TFA over 10 min), singlepeak retention time=4.01 min on 250 mm column, (M+H)+=719, (M+Na)+=741.UV/vis (100 μM in PBS) λ_(abs)=437 nm. Fluorescence (100 nM) λex=437 nm,λ_(em)=559 nm.

Example 10: 3,6-diamino-N²,N⁵-bis(26-oxo-2,5,8,11,14,17,20,23-octaoxa-27-azanonacosan-29-yl) pyrazine-2,5-dicarboxamide

To a solution of Example 7 (50.3 mg, 0.10 mmol) in DMF (5 mL) was addedTEA (109 mg, 1.08 mmol) and 2,5-dioxopyrrolidin-1-yl2,5,8,11,14,17,20,23-octaoxahexacosan-26-oate (128 mg, 0.25 mmol) andthe reaction was stirred for 16 h at room temperature. The reaction wasconcentrated and the residue was purified by medium pressure reversedphase chromatography (LiChroprep RP-18 Lobar (B) 25×310 mm—EMD chemicals40-63 μm, ˜70 g, 90/10 to 80/20 0.1% TFA-ACN) to afford 87.9 mg (82%yield) of Example 10 as an orange film: ¹NMR (300 MHz, DMSO-d6) δ 8.46(t, J=5.7 Hz, 2H), 7.96 (t, J=5.4 Hz, 2H), 3.16-3.73 (complex m, 74 H),2.28-2.32 (m, 2H). ¹³C NMR (75 MHz, DMSO-d₆) multiple conformations, δ170.1 (s), 169.9 (s) 169.8 (s), 165.1 (s), 146.0 (s), 126.2 (s). 71.2(t), 69.7 (t), 69.6 (t), 69.5 (t), 66.7 (t), 58.0 (q), 38.2 (t), 36.2(t). LCMS (15-95% gradient acetonitrile in 0.1% TFA over 10 min), singlepeak retention time=5.90 min on 250 mm column, (M+H)+=l 071,(M+2H)2+=536. UV/vis (100 μM in PBS) λ_(abs)=438 nm. Fluorescence (100nM) λex=438 nm, λ_(em)=560 nm.

Example 11: 3,6-diamino-N²,N⁵-bis(38-oxo-2,5,8,11,14,17,20, 23,26,29,32,35-dodecaoxa-39-azahentetracontan-41-y) pyrazine-2,5-dicarboxamide

To a solution of Example 7 (53.1 mg, 0.10 mmol) in DMF (5 mL) was addedTEA (114 mg, 1.13 mmol) and 2,5-dioxopyrrolidin-1-yl2,5,8,11,14,17,20,23,26,29,32, 35-dodeca-oxaoctatriacontan-38-oate (144mg, 0.21 mmol) in DMF (2.0 mL) and the resulting mixture was stirred for16 h thereafter. The reaction was concentrated and the residue waspurified by medium pressure reversed phase chromatography (LiChroprepRP-18 Lobar (B) 25×310 mm—EMD chemicals 40-63 (mm, ˜70 g, 90110 to 80/200.1% TFA-ACN) to afford 87.5 mg (61% yield) of Example 11 as an orangefilm: ¹NMR (300 MHz, DMSO-d₆) δ 8.48 (t, J=5.7 Hz, 2H), 7.96 (t, J=5.4Hz, g0 2H), 7.80-7.86 (m, 2H), 5.94 (bm, 2H), 3.30-3.60 (complex m, 106H), 2.26-2.33 (m, 4H). ¹³C NMR (75 MHz, DMSO-d₆) δ 170.2 (s), 165.1 (s),146.0 (s), 126.2 (s), 71.2 (t), 69.7 (t), 69.6 (t), 69.5 (t), 66.7 (t),58.0 (q), 38.2 (t), 36.2 (t). LCMS (15-95% gradient acetonitrile in 0.1%TFA over 10 min), single peak retention time=5.90 min on 250 mm column,(M+2H)2+=712. UV/vis (100 μM in PBS) λab=449 nm. Fluorescence (100 nM)λex=449 nm, λ_(em)=559 nm.

Example 12: 3,6-diamino-N²,N⁵-bis(2-(PEG-5000) aminoethyl)pyrazine-2,5-dicarboxamide

A solution of Example 7 (25 mg, 0.049 mmol) in DMF (30 mL) was treatedwith TEA (1 mL) and m-PEG5000-NHS (1 g, 0.2 mmol) and the resultingmixture was stirred for 48 h at room temperature. The mixture wasconcentrated and the residue was partially purified by gel filtrationchromatography (G-25 resin, water). The product was concentrated andfurther purified by reverse phase medium pressure chromatography (C18,10-70% manual gradient acetonitrile in 0.1% TFA) to afford 137.8 mg (54%yield) of Example 12 as a tan waxy solid: Maldi MS m/z=11393.

Example 13:(R)-2-(6-(bis(2-methoxyethyl)amino)-5-cyano-3-morpholinopyrazine-2-carboxamido)succinic acid

Step 1. Synthesis of 2-amino-5-bromo-3,6-dichloropyrazine

A solution of 2-amino-6-chloropyrazine (25 g, 193.1 mmol) in MeOH (500mL) was treated with NBS (34.3 g, 193.1 mmol), portion-wise, over 1hour. The resulting mixture was stirred for 16 hours thereafter. TLCanalysis at this time shows a small amount of starting materialremaining. Another 1.4 g NBS added and reaction heated to 50° C. for 2hours. The mixture was then cooled to 38° C. and treated with NCS (25.8g, 193.1 mmol). The reaction mixture was heated to 50° C. for 16 hoursthereafter. The mixture was then cooled to room temperature and treatedwith water (500 mL). The precipitate was collected by filtration anddried in a vacuum dessicator to afford 45.4 g (97% yield) of2-amino-5-bromo-3, 6-dichloropyrazine as a white solid: ¹³C NMR (75 MHz,CDCl₃) 8 149.9 (s), 145.6 (s), 129.6 (s), 121.5 (s). LCMS (15-95%gradient acetonitrile in 0.1% TFA over 10 min), single peak retentiontime=4.51 min on 30 mm column, (M+H)+=244, (M+H+ACN)+=285.

Step 2. Synthesis of 5-amino-3,6-dichloropyrazine-2-carbonitrile

A mixture of CuCN (8.62 g, 96.3 mmol) and NaCN (4.72 g, 96.3 mmol) washeated under high vacuum to 90° C. The resulting mixture was subjectedto three Argon/vacuum cycles and placed under a final positive pressureof Argon. The mixture was allowed to cool to room temperature and DMF(150 mL) was added. The heterogeneous mixture was heated to 130° C. for2.5 hours. To the resulting homogeneous mixture of sodium dicyanocupratewas added a solution of the product from Step 1 (15.6 g, 64.2 mmol)dissolved in DMF (150 mL), dropwise, over 1 hour. The temperature wasgradually raised to 150° C. and the resulting mixture was stirred atthis temperature for 10 hours thereafter. The reaction was then allowedto cool to room temperature and poured into water (1 L). The resultingmixture was extracted with EtOAc (3×) and the combined extracts werefiltered to remove a flocculent dark solid, washed with brine, dried(Na₂SO₄), filtered again and concentrated. Purification by flash columnchromatography (SiO₂, 10/1 hexanes-EtOAc to 3/1) to afford 6.70 g (55%yield) of the nitrile product as a tan solid: ¹³C NMR (75 MHz, CDCl₃) δ153.9 (s), 149.1 (s), 131.7 (s), 115.4 (s), 111.0 (s). GCMS (Inj.temperature=280° C., 1.0 mL/min helium flow rate, temperature program:100° C. (2 min hold), ramp to 300° C. @10° C./min (2 min hold), majorpeak retention time=6.56 min, m/z (EI)=188, 190.

Step 3. Synthesis of 5-amino-3-(bis(2-methoxyethyl)amino)-6-chloropyrazine-2-carbonitrile

To the product from Step 2 (1.00 g, 5.29 mmol) in ACN (20 mL) was addedbis(2-methoxyethyl)amine (3.0 mL, 2.71 g, 20.3 mmol) and the reactionmixture was heated to 70° C. for 16 hours thereafter. The reaction wascooled and concentrated. The residue was partitioned with EtOAc andwater. The organic layer was separated and the aqueous was extractedagain with EtOAc. The combined organic extracts were washed with brine,dried (Na₂SO₄), filtered and concentrated. Purification by flash columnchromatography (SiO₂, 10/1 hexanes-EtOAc to 1/1) afforded 950 mg (63%yield) of the desired adduct as a yellow solid: ¹NMR (300 MHz, CDCl₃) δ7.47 (bs, 2H), 3.77 (t, J=5.7 Hz, 4H), 3.52 (t, J=5.4 Hz, 4H), 3.25 (s,6H). ¹³C NMR (75 MHz, CDCl₃) 8 154.7 (s), 152.0 (s), 120.9 (s), 119.5(s), 95.8 (s), 71.0 (t), 59.1 (q), 50.0 (t). LCMS (50-95% gradientacetonitrile in 0.1% TFA over 10 min), single peak retention time=4.91min on 250 mm column, (M+H)+=286, (M+Na)+=308, (M+Na+ACN)+=349.

Step 4. Synthesis of3-(bis(2-methoxyethyl)amino)-5-bromo-6-chloropyrazine-2-carbonitrile

To the product from Step 3 (1.39 g, 4.88 mmol) in 48% hydrobromic acid(20 mL) at 0° C. (ice-salt bath), was added a solution of sodium nitrite(673 mg, 9.75 mmol) in water (10 mL) dropwise over 30 min. The resultingmixture concentrated. Purification by flash column chromatography (SiO₂,50/1 CHCl₃-MeOH) afforded 1.00 g (58% yield) of the bromide as anorange-brown solid: ¹NMR (300 MHz, CDCl₃) δ 3.99 (t, J=5.4 Hz, 4H), 3.64(t, J=5.4 Hz, 4H), 3.35 (s, 6H). ¹³CNMR (75 MHz, CDCl₃) δ 152.8 (s),140.8 (s), 133.4 (s), 117.2 (s), 108.3 (s), 70.4 (t), 59.1 (t), 50.5(q). LCMS (50-95% gradient acetonitrile in 0.1% TFA over 10 min), singlepeak retention time=4.55 min on 250 mm column, (M+H)+=349, 351.xture wasstirred at 0-5° C. for 1 h and poured into a stirred solution of CuBr₂(1.64 g, 7.34 mmol) in water (100 mL). The resulting mixture 45 wasstirred for 16 h at room temperature thereafter. The mixture wasextracted with EtOAc (3×). The combined organic layers were dried(Na₂SO₄), filtered and concentrated. Purification by flash columnchromatography (SiO₂, 50/1 CHCl₃-MeOH) afforded 1.00 g (58% yield) ofthe bromide as an orange-brown solid: ¹NMR (300 MHz, CDCl₃) δ 3.99 (t,J=5.4 Hz, 4H), 3.64 (t, J=5.4 Hz, 4H), 3.35 (s, 6H). ¹³CNMR (75 MHz,CDCl₃) δ 152.8 (s), 140.8 (s), 133.4 (s), 117.2 (s), 108.3 (s), 70.4(t), 59.1 (t), 50.5 (q). LCMS (50-95% gradient acetonitrile in 0.1% TFAover 10 min), single peak retention time=4.55 min on 250 mm column,(M+H)+=349, 351

Step 5. Synthesis of3-(bis(2-methoxyethylamino)-6-chloro-5-(furan-2-yl)pyrazine-2-carbonitrile

A mixture of the product from Step 4 (1.0 g, 2.87 mmol), 2-furanboronicacid (643 mg, 5.75 mmol), Cs₂CO₃ (3.31 g, 10.2 mmol), TFP (35 mol %, 236mg, 1.02 mmol), and Pd2dba3-CHCl₃ (5 mol %, 10 mol % Pd, 150 mg) wassubjected to 3 vacuum/Argon cycles and placed under a positive pressureof Argon. Anhydrous dioxane (50 mL) was added and the reaction mixturewas heated to 75° C. for 16 h thereafter. The reaction mixture wascooled to room temperature, diluted with EtOAc (100 mL) and filteredthrough a medium frit. Concentration and purification of the residue byflash chromatography (SiO₂, 50/1 CHCl₃-MeOH) afforded the 757 mg of thefuran adduct (78% yield) as a tan powder: LCMS (5-95% gradientacetonitrile in 0.1% TFA over 10 min), single peak retention time=6.41min on 250 mm column, (M+H)+=337.

Step 6. Synthesis of6-(bis(2-methoxyethyl)amino)-3-chloro-5-cyanopyrazine-2-carboxylic acid

To a well stirred mixture of ACN (11 mL), CCl₄ (7 mL), and water (11 mL)were added sodium periodate (1.07 g, 5.00 mmol) and RuO_(2.)H₂O (13.3mg, 0.10 mmol), sequentially. The resulting mixture was stirredvigorously at room temperature for 30 min and treated with sodiumbicarbonate (2.10 g, 25.0 mmol) followed by water (5 mL). Vigorousstirring for another 15 minutes was followed by the addition of asolution of the product from Step 5 (276 mg, 0.82 mmol) dissolved in ACN(1 mL). The green mixture was stirred at room temperature for 5.5 h. Themixture was transferred to a separatory funnel and extracted with EtOAc.The aqueous layer was adjusted to pH about 3.5 and extracted again withEtOAc (2×). The combined extracts were washed with 20% sodium bisulfiteand brine and dried (Na₂SO₄). Filtration and concentration afforded 140mg (54% yield) of carboxylic acid as a pale yellow solid: LCMS (5-95%gradient acetonitrile in 0.1% TFA over 10 min), single peak retentiontime=5.05 min on 250 mm column, (M+H)+=315.

Step 7. Synthesis of (R)-dibenzyl 2-(6-(bis(2-methoxy-ethyl)amino)-3-chloro-5-cyanopyrazine-2-carboxamido) succinate

A mixture of the product from step 6 (140 mg, 0.45 mmol), EDC-HCl (128mg, 0.67 mmol), HOBt H₂O (102 mg, 0.67 mmol) in anhydrous DMF (25 mL)was stirred together at room temperature for 30 min. To this stirredmixture was added (R)-dibenzyl 2-aminosuccinate TsOH salt (213 mg, 0.44mmol) followed by TEA (1 mL). The resulting mixture was stirred for 16 hthereafter. The reaction mixture was concentrated and partitioned withEtOAc and saturated sodium bicarbonate solution. The EtOAc layer wasseparated and washed with saturated sodium bicarbonate and brine, dried(Na₂SO₄), filtered and concentrated to afford 5 240 mg (88% yield) ofthe pyrazine amide as an orange foam: LCMS (15-95% gradient acetonitrilein 0.1% TFA over 10 min), single peak retention time=8.76 min on 250 mmcolumn, (M+H)+=610, (M+Na)+=632.

Step 8. Synthesis of (R)-dibenzyl 2-(6-(bis(2-methoxyethyl)amino)-5-cyano-3-morpholinopyrazine-2-carboxamido)succinate

To the product from Step 7 (240 mg, 0.39 mmol) was added morpholine (5mL). The reaction mixture was heated to 70° C. for 2 h. The mixture wascooled and concentrated. The residue was partitioned with EtOAc andwater. The EtOAc layer was separated and washed with saturated sodiumbicarbonate and brine. The EtOAc layer was dried (Na₂SO₄), filtered andconcentrated. Purification by flash column chromatography (SiO₂, 3:1 to1:1 hexanes-EtOAc) afforded 199 mg (75% yield) of the morpholine adductas an orange foam: LCMS (15-95% gradient acetonitrile in 0.1% TFA over10 min), single peak retention time=8.76 min on 250 μm column,(M+H)+=661, (M+Na)+=683.

Step 9. Synthesis of (R)-2-(6-(bis(2-methoxyethyl)amino)-5-cyano-3-morpholinopyrazine-2-carboxamido)succinic acid

The dibenzyl ester (115 mg, 0.17 mmol) in THF (10 mL) was added 1.0 Nsodium hydroxide (4 mL). The mixture was stirred for 1 h at roomtemperature. The pH was adjusted to about 2 with 1.0 N HCl and thesolution was concentrated. Purification of the residue by mediumpressure reversed phase chromatography (LiChroprep RP-18 Lobar (B)25×310 mm—EMD chemicals 40-63 (μm, ˜70 g, 90/10 to 50/500.1% TFA-ACN)afforded 32 mg (27% yield) of Example 13 as an orange solid: LCMS(15-95% gradient acetonitrile in 0.1% TFA over 10 min), single peakretention time=4.47 min on 250 mm column, (M+H)=481. UV/vis (100 μM inPBS) λ_(abs)=438 nm. Fluorescence (100 μM) λex=449 nm, λ_(em)=570.

Example 14: 3,6diamino-N²,N⁵-bis(2-hydroxy-β-serine)-pyrazine-2,5-dicarboxamide

The title compound may be prepared by using substantially the procedureof Example 2 but substituting 2-OBn-β-serine-OMe-HCl salt for(D)-Ser(OBn)-OMe-HCl salt in an equivalent amount in Step 1.

Example 15: 3,6-diaminopyrazine-2,5-dicarboxylic acid

Dipotassium 2,4,6,8-tetrahydroxypyrimido(4,5-g)pteridine was prepared bytreating 5-aminouracil with potassium ferricyanide in the presence ofpotassium hydroxide as described in Taylor et al., JACS, 77: 2243-2248(1955).

In each of two Teflon reaction vessels was placed 0.5 g dipotassium2,4,6, 8-tetrahydroxypyrimido[4,5 g]pteridine and a solution consistingof 0.3-0.4 g sodium hydroxide in about 10 mL deionized water. Thevessels were secured in the microwave reactor and allowed to react forone hour at 170° C., generating ca. 100 psig pressure, for one hour. Thevessels were allowed to cool in the microwave to ca. 50° C. and thecontents filtered to remove a small amount of solid residue. The brightyellow filtrate was transferred to a 250 mL round-bottom flask equippedwith a large magnetic stir bar. With stirring, the pH was adjusted toca. 3 with concentrated HCl. A large amount of red precipitate formed. Afew more drops of acid was added and the solid collected by filtrationon a glass frit, washed with cold 1N HCl (1×10 mL), acetonitrile (×30mL) and diethyl ether (1×30 mL), suctioned dry and transferred to avacuum oven, vacuum drying overnight at 45-50° C. Yield 0.48 g (79%).C¹³ NMR (D₂O/NaOD, external TMS reference) 6 132.35, 147.32, 171.68.

Example 16:3,6-diamino-N²,N⁵di(2,5,8,11,14,17,20,23,26,29,32,35,38,41,44,47,50,53,56,62,65,68,71-tetracosaoxatriheptacosan-73-yl)pyrazine-2,5-dicarboxamide

A round-bottom flask (2 L) equipped with a magnetic stir bar was chargedwith 3,6-diaminopyrazine-2,5-dicarboxylic acid (3.50 g, 17.7 mmol),m-dPEG24-amine (45 g, 40.2 mmol), and PyBOP (21.4 g, 41.1 mmol) inanhydrous DMF (1 L) and the reaction mixture was purged with argon.Triethylamine (50 mL) was slowly added to the suspension and within anhour the reactants had dissolved leading to a dark red solution. Thereaction mixture was stirred overnight at rt, concentrated under highvacuum to give the crude product as a dark red oil that was purified byreverse phase preparative HPLC (column: Waters XBridge Prep C18 5 mm OBD30×150 mm; λ_(max); PDA (200-800 nm), flow rate: 50 mL/min; gradient:A:B 95:5 / 1 min, 50:50/8 min, 5:95/8.1 min, 5:95/10 min (A: H₂O/0.1%TFA, B: CH₃CN/0.1% TFA). Product containing fractions were combined,concentrated in vacuo and the residue dissolved in CHCl₃ (150 mL),washed with saturated NaHCO₃ (2×75 mL) and brine (75 mL), dried overNa₂SO₄ and evaporated to dryness. The gummy residue was co-evaporatedwith EtOH (absolute) and dried overnight under high vacuum at 40° C. togive the title product as a red solid (20.0 g, 61%). ¹H NMR (CDCl₃) δ8.13 (t, J=5.8 Hz, 2H), 6.06 (s, 4H), 3.68-3.54 (m, 192H), 3.38 (s, 6H);¹³C NMR (CDCl₃) δ 165.3, 146.6, 126.9 71.9, 70.63, 70.6, 70.56, 70.5,70.4, 69.8, 59.0, 39.0; RP-LC/MS (ESI) m/z 1179.1 (M+H+NH₄)²⁺(t_(r)=3.88min). Anal. Calcd for C₁₀₄H₂₀₄N₆O₅₀: C, 53.41; H, 8.79: N, 3.61. Found:C, 53.15: H, 8.81; N, 3.61.

Example 17:3,6-bis(2,5,8,11,14,17,20,23,26,29,32,35-dodecaoxaoctatriacontan-38-ylamino)-N²,N⁵-di(2,5,8,11,14,17,20,23,26,29,32,35-dodecaoxaheptatriacontan-37-yl)pyrazine-2,5-dicarboxamide

A round-bottom flask (500 mL) equipped with a magnetic stir bar wascharged with the product of Example 11,3,6-diamino-N²,N⁵-bis(38-oxo-2,5,8,11,14,17,20, 23,26,29,32,35-dodecaoxa-39-azahentetracontan-41-y) pyrazine-2,5-dicarboxamide (6.24g, 4.87 mmol) and dissolved in anhydrous 1,2-dichloroethane (DCE, 100mL) under argon. To the resulting orange solution, a solution ofm-dPEG12-propionaldchydc (7.34 g, 12.8 mmol) in DCE (25 mL) and glacialHOAc (0.73 mL, 12.7 mmol) were added in succession. To this was addedsodium triacetoxyborohydride (2.71 g, 12.8 mmol) in portions (0.50 g)over a 1.5 hr period each time rinsing the transfer vial with DCE (75 mLtotal). The resulting reddish suspension was stirred overnight at rtunder argon and the reaction was quenched by slow addition of saturatedsodium bicarbonate (100 mL). The biphasic mixture was stirred for 30min, layers separated, and the aqueous layer extracted further withCHCl₃ (50 mL). The combined organic extracts were washed with brine (100mL) and then dried over Na₂SO₄. Removal of volatiles in vacuo gave 12.8g of crude product as a red solid that was purified by reverse phasepreparative HPLC (column: Waters XBridge Prep C18 5 mm OBD 30×250 mm;λ_(max); 280 nm, flow rate: 50 mL/min; gradient: A:B 75:25/0 min,75:25/5 min, 45:55/40 min, 5:95/8.1 min, 5:95/40 min (A: H₂O/0.1% TFA,B: CH₃CN/0.1% TFA). Product containing fractions were combined,concentrated in vacuo and the residue dissolved in CHCl₃ (200 mL),washed with saturated NaHCO₃ (2×100 mL) and brine (100 mL), dried overNa₂SO₄ and evaporated to dryness. The red viscous residue wasco-evaporated with EtOH (absolute) and dried overnight under high vacuumat 40° C. to give the title product as a red brick solid (7.40 g, 63%).¹H NMR (DMSO-d₆) δ 8.42 (t, J=5.8 Hz, 2H), 7.88 (t, j=5.5 Hz, 2H),3.64-3.34 (m, 192H), 3.23 (s, 12H), 1.79-1.75 (quintet, J=6.4 Hz, 4H);¹³C NMR (DMSO-d₆) δ 165.9, 145.9, 126.3 71.7, 70.28, 70.25, 70.2, 70.12,70.09, 70.05, 69.3, 68.8, 58.5, 39.0, 38.1, 29.8; RP-LC/MS (ESI) m/z2394.5 (M+H)⁺, 1197.7 (M+2H)²⁺(t_(r)=4.09 min). Anal. Calcd forC₁₀₈H₂₁₂N₆O₅₀: C, 54.16; H, 8.92: N, 3.51. Found: C, 54.31: H, 8.91; N,3.52.

Example 18: 3,3′-((3,6-diaminopyrazine-2,5 dicarbonyl)bis(azanediyl))dipropionic acid

Step 1. Synthesis of dibenzyl 3,3′-((3,6-diaminopyrazine-2,5-dicarbonyl)bis(azanediyl)) dipropionate

A flame dried round-bottom flask (100 mL) equipped with a magnetic stirbar was charged with 3,6-diaminopyrazine-2,5-dicarboxylic acid (0.30 g,1.54 mmol), benzyl 3-aminopropanoate p-toluene sulfonate (1.08 g, 3.08mmol, MW 351.42), EDC HCl (0.590 g, 3.08 mmol, 191.7 g/mol), HOBt 3H₂O(0.582 g, 3.08 mmol, 189.13 g/mol), and Et₃N (1.50 g, 15 mmol, 101.2g/mol, 2.0 mL d 0.73 g/mL) in DMF (anhydrous, 40 mL cannula transferredto the reaction vessel using Ar). An argon atmosphere was maintainedthroughout the course of the experiment. The reaction mixture becomesbrown over time was stirred overnight at rt and concentrated in vacuo toabout 10 mL (8 mbar at 60° C. water bath). Remaining DMF was removed bytoluene azeotrope (2×10 mL toluene). The dark reaction mixture waspartitioned between EtOAc (3×125 mL) and sat'd NaHCO₃ (3×100 mL).Combined the organic layers and partitioned against citric acid (10%aqueous, 100 mL) followed by brine. The organic layer was removed, dried(Na₂SO₄ anhydrous) and concentrated in vacuo to give a reddishcrystalline solid, 0.58 g. TLC (EMD Silica gel 60 on glass support,EtOAc) shows one major spot (characteristically highly fluorescent usingblue LED light) running close to the solvent front and very littleremaining at the origin). TLC (1:1 EtOAc:hexanes) Rf 0.22. The productwas purified via flash chromatography over silica gel (Teledyne ISCOGOLD column, 24 g, gradient: 100% hexanes to 100% EtOAc over 12 min at35 mL/min with detection at both 254 nm and 450 nm) to give (0.49 g, 62%isolated yield). Mass spectrum (ES+) 521.36 (100%), 522.42 (30%), 523.34(approx. 6%). NMR, ¹H (DMSO-d₆), 400 MHz: 2.55 (4H, m), 3.41 (4H, m)5.01 (4H, s), 6.44 (4H, s), 7.21 (10H, m), 8.41 (2H, m); ¹³C (DMSO-d₆):34.18, 35.33, 66.19, 126.74, 128.52, 128.92, 136.56, 146.75, 165.63,171.90.

Step 2. Synthesis of dibenzyl 3,3′-((3,6-diaminopyrazine-2,5-dicarbonyl)bis(azanediyl)) dipropionate to3,3′-((3,6-diaminopyrazine-2,5-dicarbonyl)bis(azanediyl))dipropionicacid

The di-benzyl ester of pyrazine-di-beta-alanine (dibenzyl3,3′-((3,6-diaminopyrazine-2, 5-dicarbonyl)bis(azanediye)dipropionate)prepared in Step 1 (0.92 g) was combined with EtOH (abs., 75 mL) andtransferred to a Fischer-Porter pressure bottle (6 oz) equipped withinlet and outlet valves, a pressure gauge (0-100 psig) and a tefloncoated magnetic stir bar. To this was added water (25 mL) and 10% Pd oncarbon (0.2 g, Degussa/Aldrich wet) and the reaction vessel sealed.Following three vacuum/Ar cycles, hydrogen was introduced from a lecturebottle at 10 psig to a vigorously stirred solution. After 3.5 hrs asample was withdrawn and TLC analysis (EMD Silica gel 60 on glasssupport, EtOAc) showed no starting material present only a highly yellowfluorescence spot (excitation by blue LED) at the origin. The crudereaction was filtered through a pad of celite and the resultingcelite/catalyst bed rinsed with about 500 mL 1:1 EtOH: H₂O to obtain ayellow solution that was concentrated to a solid by rotovap (50° C.). Atotal of 0.424 g red solid was isolated (70.5% isolated yield). A smallsample (about 1 mg) was dissolved in acetonitrile: water (4:1) andexamined using a Waters Acquity UPLC using both UV and fluorescencedetectors (FLD) (elution program: 0 to 3 min; 90% A: 10% B: 3 to 20min;10% A: 90% B: 20.1 to 30 min: 90% A to 10% B. A=water plus 0.01%TFA. B=ACN plus 0.01% TFA. UV at 264 nm. Column: Phenomenex Luna C18,250 mm×4.5 mm). Only one peak was observed at 9.3 minutes. Mass spectrum(ES+) 341.32 (100%), 342.37 (30%), 344.29 (18%), 270.30 (62%). NMR, ¹H(DMSO-d₆), 400 MHz: 2.54 (2H, m), 3.42 (2H, m), 6.52 (2H, s), 7.21 (4H,m), 8.38 (2H, m), 11.9 (2H, bs); ¹³C (DMSO-d6): 34.20, 35.33, 126.77,146.75, 165.55, 173.57.

Example 19: 3,6-Diamino-N²,N⁵-bis(4-amino-butyricacid)-pyrazine-2,5-dicarboxamide

Step 1. Synthesis of 3,6-Diamino-N²,N⁵-bis(ethyl4-amino-butyrate)-pyrazine-2, 5-dicarboxamide diethyl ester

A mixture of sodium 3,6-diaminopyrazine-2,5-dicarboxylate (300 mg, 1.24mmol), (ethyl 4-amino-butyrate HCl salt (2.64 mmol), HOBt-H₂0 (570 mg,3.72 mmol) and EDC-HCI (690 mg, 3.60 mmol) in DMF (25 mL) is treatedwith TEA (2 mL). The resulting mixture is stirred for 16 h andconcentrated. The resulting mixture is concentrated to dryness and theresidue partitioned between EtOAc and water. The layers are separatedand the EtOAc solution washed with NaHCO₃ (saturated) and brine. Thesolution is dried over Na₂SO₄ (anhydrous), filtered and concentrated.Purification by radial flash chromatography or by reverse phase hplcusing a C18 column affords the desired pyrazine intermediate that iscarried forward in the next step.

Step 2. Synthesis of 3,6-Diamino-N²,N⁵-bis(4-amino-butyricacid)-pyrazine-2, 5-dicarboxamide

The product from Step 1 in THF (about 400 mg in about 10 mLs THF shouldsuffice) is treated with sodium hydroxide in water (1.0 N). Afterstirring at room temperature for about 30 min (or until the reaction isdeemed complete by TLC) the pH is adjusted to approximately 2 by theaddition of HCl in water (1.0 M) and the resulting solution extractedwith EtOAc (3×). The organic layers are combined, dried (Na₂SO₄,anhydrous), filtered, concentrated and purified by rphplc to afford thetitle compound, 3,6-Diamino-N²,N⁵-bis(4-amino-butyricacid)-pyrazine-2,5-dicarboxamide.

Example 20: 3,6-Diamino-N²,N⁵-bis(3-amino-5-oxo-furane)-pyrazine-2,5-dicarboxamide

A mixture of sodium 3,6-diaminopyrazine-2,5-dicarboxylate (300 mg, 1.24mmol), 3-amino-5-oxo-furane HCl salt (synthesized as described in U.S.Pat. No. 6,037,365) (2.64 mmol), HOBt-H₂0 (570 mg, 3.72 mmol) andEDC-HCI (690 mg, 3.60 mmol) in DMF (25 mL) is treated with triethylamine(2 mL). The resulting mixture is stirred for 16 h and concentrated todryness and the residue taken up in the minimum amount of water. Theaqueous mixture is acidified with TFA to pH about 2. Purification isachieved by reverse phase HPLC using a C18 column and awater:acetonitrile gradient to afford the desired pyrazine bis-lactone.

Example 21: 3,6-Diamino-N²,N⁵bis (2,3-dimethyl-beta-alanine)-pyrazine-2,5-dicarboxamide

Step 1. Synthesis of 3,6-Diamino-N²,N⁵-bis (ethyl-2,3-dimethyl-beta-alanine)-pyrazine-2, 5-dicarboxamide

A mixture of sodium 3,6-diaminopyrazine-2,5-dicarboxylate (300 mg, 1.24mmol), 2, 3-dimethyl-beta-alaninc ethyl ester HCl salt (2.64 mmol),HOBt-H₂O (570 mg, 3.72 mmol) and EDC-HCI (690 mg, 3.60 mmol) in DMF (25mL) is treated with triethylamine (2 mL). The resulting mixture isstirred at room temperature for 16 h and concentrated. The resultingmixture is concentrated to dryness and the residue partitioned betweenEtOAc and water. The layers are separated and the EtOAc solution washedwith NaHCO₃ (saturated) and brine. The resulting organic solution isdried over Na/SO₄ (anhydrous), filtered and concentrated. Purificationby radial flash chromatography, normal medium pressure flashchromatography or by reverse phase HPLC using a C18 column affords thedesired pyrazine intermediate that is carried forward in the next step.

Step 2. Synthesis of 3,6-Diamino-N²,N⁵-bis (2,3-dimethyl-beta-alanine)-pyrazine-2, 5-dicarbox amide

The product diethyl ester from Step 1 in THF (about 400 mg in about 10mL should suffice) is treated with sodium hydroxide in water (1.0 N).After stirring at room temperature for about 30 min (or until thereaction is deemed complete by TLC) the pH is adjusted to approximately2 by the addition of HCl in water (1.0 M) and the resulting solutionextracted with EtOAc (3×). The organic layers are combined, dried(Na₂SO₄, anhydrous), filtered, and concentrated filtered to afford3,6-Diamino-N²,N⁵bis (2,3-dimethyl-beta-alanine)-pyrazine-2,5-dicarboxamide that may be purified by RPHPLC using a C18 column usingan appropriate gradient of acetonitrile in 0.1% TFA and water in 0.1%TFA.

Example 22: 3,6-Diamino-N²,N⁵-bis (3-(3-pyridyl)-propionicacid)-pyrazine-2, 5-dicarboxamide

Step 1. Synthesis of 3,6-Diamino-N²,N⁵-bis (ethyl3-(3-pyridyl)-propanoate)-pyrazine-2, 5-dicarboxamide

A mixture of sodium 3,6-diaminopyrazine-2,5-dicarboxylate (300 mg, 1.24mmol), (ethyl 3-(3-pyridyl)-propanoate HCl salt (2.64 mmol) (J. G. Rico,et.al., J. Org. Chem., 1993, 58, 7948-7951), HOBt-H₂O (570 mg, 3.72mmol) and EDC-HCI (690 mg, 3.60 mmol) in DMF (25 mL) is treated withtriethylamine (2 mL). The resulting mixture is stirred for 16 h at roomtemperature and concentrated. The resulting mixture is dissolved in theminimum amount of water, acidified to about pH 3 by addition of TFA andpurified by by reverse phase HPLC using a C18 column affording thedesired pyrazine intermediate diethyl ester that is carried forward inthe next step.

Step 2. Synthesis of 3,6-Diamino-N²,N⁵-bis (ethyl3-(3-pyridyl)-propanoate)-pyrazine-2, 5-dicarbox amide

The product from Step 1 in THF (about 400 mg in about 10 mLs THF) istreated with sodium hydroxide or lithium hydroxide in water (1.0 N).After stirring at room temperature for about 30 min (or until thereaction is deemed complete by TLC) the pH is adjusted to approximately2 by the addition of TFA and the resulting solution purified by RPHPLC(C18, acetonitrile 0.1% TFA and water 0.1% TFA gradient) to afford thetitle compound, 3, 6-Diamino-N²,N⁵-bis (ethyl3-(3-pyridyl)-propanoate)-pyrazine-2,5-dicarboxamide.

Example 23: (2R,2′R)-2,2′-((3,3′-((3,6-diaminopyrazine-2,5-dicarbonyl)bis(azanediyl))bis(propanoyl))bis(azanediyl))disuccinicacid

Step 1. Synthesis of tetrabenzyl 2,2′-((3,3′-((3,6-diaminopyrazine-2,5-dicarbonyl)bis(azanediyl))bis(propanoyl))bis(azanediyl))(2R,2′R)-disuccinate

3,3′-((3,6-diaminopyrazine-2,5 dicarbonyl)bis(azanediyl)) dipropionicacid produced in Example 18 (0.50 g, 1.47 mmol) is treated with DMF (30mL) in a three-necked round bottom flask (100 mL), equipped with ateflon coated magnetic stir bar and gas inlet and outlet (Argon, dry).To the mixture is added Asp (OBz)₂ p-TSA(1.57 g, 3.23 mmol, 2.2 equiv),DIPEA (0.45 g, 3.5 mmol), and PyBop (1.67 g, 3.2 mmol) and the mixtureallowed to stir overnight. The reaction mixture is concentrated undervacuum by rotavap and purified by normal phase chromatography oversilica gel using a Combi-Flash R_(f) Unit (Teledyne ISCO) andhexanes-EtOAc gradiant. Fractions containing the desired tetra-benzylester are combined and concentrated to a solid, substantially puretetrabenzyl2,2′-((3,3′-(3,6-diaminopyrazine-2,5-dicarbonyl)bis(azanediyl))bis(propanoyl))bis(azanediyl))(2R,2′R)-disuccinate is obtained and usedin the next step.

Step 2. Synthesis of (2R,2′R)-2,2′-((3,3′-((3,6-diaminopyrazine-2,5-dicarbonyl)bis(azanediyl))bis(propanoyl))bis(azanediyl))disuccinicacid

Tetrabenzyl2,2′-((3,3′-((3,6-diaminopyrazine-2,5-dicarbonyl)bis(azanediyl))bis(propanoyl)) bis(azanediyl))(2R,2′R)-disuccinate (0.5 g, 0.88 mmol)prepared in Step 1 is placed in a Fischer-Porter pressure bottle (6 oz)equipped with a Teflon covered magnetic stirring bar and dissolved inEtOH (20 mL). Catalyst (0.1 g 5% Pd on carbon) is added and vigouousstirring commenced. After several Ar/vacuum cycles, hydrogen is addedand the reaction carried out in a similar fashion to Example 18 anddesired product, (2R,2′R)-2,2′-((3,3′-((3,6-diaminopyrazine-2,5-dicarbonyl)bis(azanediyl))bis(propanoyl))bis(azanediyl)) disuccinic acid, obtained.

Example 24: Sodium3,3′-((3,6-diaminopyrazine-2,5-dicarbonyl)bis(azanediyl))bis(4-hydroxybutanoate)

The product di-lactone isolated in Example 20 is dissolved in water(deionized) and two equivalents of base, sodium hydroxide, for example,dissolved in water added with stirring forming the desired di-hydroxyacid sodium salt, sodium 3,3′-((3,6-diaminopyrazine-2,5-dicarbonyl)bis(azanediyl))bis(4-hydroxybutanoate). Solid product maybe obtained by removing residual water by distillation or by freezedrying (lyophilizing) the reaction mixture.

Example 25: (2R,2′R)-3,3′-((3,6-diaminopyrazine-2,5-dicarbonyl)bis(azanediyl))bis(2-(hydroxymethyl)propanoic acid)

Step 1. Synthesis of dimethyl3,3′-((3,6-diaminopyrazine-2,5-dicarbonyl)bis(azanediyl))(2R,2′R)-bis(2-((benzyloxy)methyl)propanoate)

A mixture of sodium 3,6-diaminopyrazine-2,5-dicarboxylate (300 mg, 1.24mmol), methyl-2-O-benzyl-3-amino-propionate HCl salt (2.64 mmol),HOBt-H₂0 (570 mg, 3.72 mmol) and EDC-HCI (690 mg, 3.60 mmol) in DMF (25mL) is treated with triethylamine (2 mL). The resulting mixture isstirred for 16 h, concentrated, and partitioned between EtOAc and water.The layers are separated and the EtOAc layer washed with NaHCO₃(saturated) and brine. The organic layer is dried over Na₂SO₄(anhydrous), filtered and concentrated. Purification by radial flashchromatography (silica) or by reverse phase HPLC using a C18 columnaffords the desired pyrazine intermediate that is carried forward in thenext step.

Step 2. Synthesis of (2R,2′R)-3,3′-((3,6-diaminopyrazine-2,5-dicarbonyl)bis(azanediyl))bis(2-((benzyloxy)methyl)propanoic acid)

The product from Step 1 in THF (about 400 mg in about 10 mLs THF) istreated with sodium hydroxide in water (1.0 N). After stirring at roomtemperature for about 30 min (or until the reaction is deemed completeby TLC) the pH is adjusted to approximately 2 by the addition of HCl inwater (1.0 M) and the resulting solution extracted with EtOAc (3×). Theorganic layers are combined, dried (Na₂SO₄, anhydrous), filtered andconcentrated to afford the diacid.

Step 3. Synthesis of (2R,2′R)-3,3′-((3,6-diaminopyrazine-2,5-dicarbonyl)bis(azanediye)bis(2-(hydroxymethyl)propanoic acid)

To the product of Step 2 in methanol is added 5% Pd/C and ammoniumformate (about a 10 fold molar excess over diacid) and the resultingsolution heated to reflux for about 2 hours. The reaction is cooled toroom temperature, filtered and concentrated. The residue may be purifiedeither by recrystallizing from an appropriate solvent or by reversephase HPLC (for example, using a C18 column and a 5-95% gradientacetonitrile in 0.1% TFA and water in 0.1% TFA over about 10 to 30 mins)to obtain the title compound in substantially pure form.

Example 26: Assessing Vasculature in Mice

An exemplary procedure for assessing vasculature is as follows: Allexperiments will be performed on anesthetized mice. A Maestro in vivoimaging system from CRi or equivalent will be employed. Commercialfluorescein solution for angiography will be obtained and used as is.Pyrazine dyes in a physiological compatible solution (phosphate bufferedsolution) will be supplied. During the experiment the mouse will bepositioned on a thermostated water blanket, with body temperaturemonitored by a rectal temperature probe. To facilitate imaging of thevessels of interest, the thoracic, abdominal and inguinal areas of themouse will be shaved, the mouse positioned on its back and the skin overthe femoral vasculature resected to expose the vasculature of interest.The jugular vein will be cannulated using a piece of stretched PE10tubing filled with saline containing 50 U heparin/mL. After the mouse isprepared a bolus injection of dye is administered followed by an i.v.bolus of saline solution. All injections are administered via thecannula established in the jugular vein. The saline solution is used toflush the line and ensure passage of an intact bolus through the femoralvasculature, producing a sharp wavefront.

An exemplary procedure for assessing eye vasculature is as follows: Allexperiments will be performed on anesthetized mice. Retinal images willbe taken using a Phoenix Micron III Retinal Imaging Microscope.Commercial fluorescein solution for eye angiography will be obtained andused as is. The pyrazine dye in a physiological compatible solution(phosphate buffered solution) will be supplied by MediBeacon, LLC.Administration of each agent will be by typical tail vein injection.

Two mice designated 1 and 2 will be evaluated using the fluoresceinsolution initially. A pre-administration angiogram will be takenfollowed by administration of the standard fluorescein solution.Angiograms will be taken immediately after administration and then at 5,10, 30, and 60 minutes post-administration.

Two mice designated 3 and 4 will be evaluated using the pyrazineformulation initially. A pre-administration angiogram will be takenfollowed by administration of the standard fluorescein solution.Angiograms will be taken immediately after administration and then at 5,10, 30, and 60 minutes post-administration.

At least 24 hours after the initial experiments on mice 1, 2, 3, and 4,repeat the procedure with mice 1 and 2 receiving the pyrazineformulation and mice 3 and 4 receiving the fluorescein solution.Comparison of all images will be performed.

Example 27: Comparison of Fluorescence Angiography Using Example 2 andFluorescein In Vivo

In a proof of concept study to establish that Example 2(3,6-diamino-N2,N5-bis (D-serine)-pyrazine-2,5-dicarboxamide) can beused to visualize ocular vasculature, retinal fluorescence was imagedusing a Phoenix Micron III Retinal Imaging Microscope followinginjection of fluorescein and Example 2 in separate, individual mice.Example 2 and fluorescein (both dosed at 1 mg per mouse) was injected inthe femoral vein of six week old male C57B1/6J mice and retinal imageswere obtained at various times following injection. FIG. 1 shows theimages obtained at 2 minutes. Similar structural detail is observed withExample 2 as with fluorescein. Due to the development of cataracts inresponse to the imaging, longitudinal observation and within animalcomparison of fluorescein and Example 2 was not possible.

Example 28: Intraoperative Fluorescence Angiography Assessing CerebralPerfusion Patients and Subjects

Patients undergoing STA-MCA bypass surgery for unilateral major cerebralartery occlusive disease would be enrolled in the study. Indications forSTA-MCA bypass surgery for impaired cerebral perfusion would bedetermined according to the study criteria. In all surgeries, both thefrontal and parietal branches of the STA would be anastomosed to thebranches of the MCA in an end-to-side fashion. Patients would completethe PET protocol within 1 month before surgery and complete theintraoperative near-infrared ICG videoangiography (ICG-VA) or3,6-diamino-N²,N⁵-bis(D-serine)-pyrazine-2,5-dicarboxamide protocolduring surgery. Following surgery, systolic blood pressure would bestrictly controlled between 100 and 140 mmHg, and anticonvulsantmedication would be administered intravenously. Using single photonemission computed tomography with technetium-99m hexamethylpropyleneamine oxime (99mTc-HM-PAO SPECT), cerebral blood flow (CBF) measurementswould be taken at 1 and 7 days after surgery. The postoperative state ofthe brain and the patency of bypass would also be assessed by magneticresonance imaging (MRI)/magnetic resonance angiography (MRA) at 1 and 7days after surgery. If it was suspected that patient presented withsymptoms associated with hyperperfusion, evaluation by SPECT and MRI/MRAwould be performed.

Patients undergoing craniotomy and clipping surgery for unrupturedcerebral aneurysms would serve as control subjects. These subjectspresenting with no steno-occlusive disease, as assessed by intracranialMRA and neck MRA/neck ultrasonography, would complete the ICG-VA or3,6-diamino-N²,N⁵-bis(D-serine)-pyrazine-2,5-dicarboxamide protocol justafter fronto-temporal craniotomy.

The institutional medical review board would approve the study protocoland all patients would provide written informed consent.

ICG-VA and 3,6-diamino-N²,N⁵-bis(D-serine)-pyrazine-2,5-dicarboxamideProtocol

The recommended dose of ICG-VA is 0.1-0.3 mg/kg, and the daily dosewould not exceed 5 mg/kg. In this series or arm of the study, patientswould complete the ICG-VA study protocol just after fronto-temporalcraniotomy, and the patients undergoing bypass surgery would completethe same protocol just after bypass procedure. Subjects would receive astandard dose of 7.5 mg per injection dissolved in 3.0 ml of physiologicsaline. The recording would commence and a calculated bolus of ICG wouldbe administered by the anesthesiologist at the surgeon's request. TheICG transit curves intensities would be recorded by an automaticmicroscope-integrated algorithm using near-infrared light (λ=800 nm;OPMT Pentero microscope with infrared fluorescence detection hardwareand the Flow 800 software analysis tool; Carl Zeiss Meditec, Oberkochen,Germany). This tool features an algorithm for correcting shading andbrain pulsation. Fluorescence intensities would be measured in arbitraryintensity units (AIs) that corresponded to the intensity detected by thecamera. The additional time needed for ICG angiography was approximately90 s. Normal cardiac function (ejection fraction >55%) would also beconfirmed preoperatively in all patients.

Patients in the3,6-diamino-N²,N⁵-bis(D-serine)-pyrazine-2,5-dicarboxamide (Example 2)arm of the study would receive the appropriate dose of pyrazine dye inphysiologic saline buffer and would complete the pyrazine dye protocoljust after fronto-temporal craniotomy and the patients undergoing bypasssurgery would complete the same protocol just after bypass surgery,receiving the standard dose prescribed for the pyrazine dye. Data forthis arm would be obtained as outlined in the previous paragraph forICG.

The course of fluorescent intensities would be measured by freelydefinable regions of interest (ROIs). The data from ROIs would beexported for further processing after surgery and ICG results would becompared to those obtained by the pyrazine dye.

The following compounds in can generally be made using the methodsdescribed above. It is expected that these compounds when made will haveactivity similar to those that have been prepared.

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Other Embodiments

The detailed description set-forth above is provided to aid thoseskilled in the art in practicing the present disclosure. However, thedisclosure described and claimed herein is not to be limited in scope bythe specific embodiments herein disclosed because these embodiments areintended as illustration of several aspects of the disclosure. Anyequivalent embodiments are intended to be within the scope of thisdisclosure. Indeed, various modifications of the disclosure in additionto those shown and described herein will become apparent to thoseskilled in the art from the foregoing description, which do not departfrom the spirit or scope of the present inventive discovery. Suchmodifications are also intended to fall within the scope of the appendedclaims.

1-29. (canceled)
 30. A method of assessing the location of a disease or an injury in a subject's vasculature, comprising the steps of: a. administering an effective amount of a compound of structural Formula I

 or a salt thereof; wherein X¹ and X² are independently chosen from —CO(AA), —CN, —CO₂R¹, —CONR²R³, —COR⁴, —NO₂, —SOR³⁵, —SO₂R⁶, —SO₂OR⁷ and —PO₃R⁸R⁹; Y¹ and Y² are independently chosen from OR¹⁰, —SR¹¹, —NR¹²R¹³, —N(R¹⁴)COR¹⁵, —CONH(PS): —P(R¹⁶)₂, —P(OR¹⁷)₂ and

 each Z¹ is independently chosen from a bond, —CR¹⁸R¹⁹, —O—, —NR²⁰—, —NCOR²¹—, —S—, —SO—, and —SO₂—; each R¹ to R²¹ are independently chosen from hydrogen, C₁-C₁₀ alkyl optionally substituted with hydroxyl and carboxylic acid, C₃-C₆ polyhydroxylated alkyl, C₅-C₁₀ aryl, C₅-C₁₀ heteroaryl, C₃-C₅ heterocycloalkyl optionally substituted with C(O), —(CH₂)_(a)CO₂H optionally substituted with C₅-C₁₀ heteroaryl, —(CH₂)_(a)CONR³⁰R³¹, —(CH₂)_(a)NHSO₃ ⁻, —(CH₂)_(a)NHSO₃H, —(CH₂)_(a)OH, —(CH₂)_(a)OPO₃ ⁼, —(CH₂)_(a)OPO₃H₂, —(CH₂)_(a)OPO₃H⁻, —(CH₂)_(a)OR²², —(CH₂)_(a)OSO₃ ⁻, —(CH₂)_(a)OSO₃H, —(CH₂)_(a)PO₃ ⁼, —(CH₂)_(a)PO₃H₂, —(CH₂)_(a)PO₃H⁻, —(CH₂)_(a)SO₃ ⁻, —(CH₂)_(a)SO₃H, —(CH₂)_(d)CO(CH₂CH₂O)_(c)R²³, —(CH₂)_(d)(CH₂CH₂O)_(c)R²⁴, —(CHCO₂H)_(a)CO₂H, —CH₂(CHNH₂)_(a)CH₂NR²⁵R²⁶, —CH₂(CHOH)_(a)CO₂H, —CH₂(CHOH)_(a)R²⁷, —CH[(CH₂)_(b)NH₂]_(a)CH₂ OH, —CH[(CH₂)_(b)NH₂]_(a)CO₂H, and —(CH₂)_(a)NR²⁸R²⁹; each R²² to R³¹ are independently chosen from hydrogen, C₁-C₁₀ alkyl, and C₁-₅-dicarboxylic acid; R³⁵ is chosen from C₁-C₁₀ alkyl optionally substituted with hydroxyl and carboxylic acid, C₃-C₆ polyhydroxylated alkyl, C₅-C₁₀ aryl, C₅-C₁₀ heteroaryl, C₃-C₅ heterocycloalkyl optionally substituted with C(O), —(CH₂)_(a)CO₂H optionally substituted with C₅-C₁₀ heteroaryl, —(CH₂)_(a)CONR³⁰R³¹, —(CH₂)_(a)NHSO₃ ⁻, —(CH₂)_(a)NHSO₃H, —(CH₂)_(a)OH, —(CH₂)_(a)OPO⁼, —(CH₂)_(a)OPO₃H₂, —(CH₂)_(a)OPO₃H⁻, —(CH₂)_(a)OR²², —(CH₂)_(a)OSO₃ ⁻, —(CH₂)_(a)OSO₃H, —(CH₂)_(a)PO₃ ⁻, —(CH₂)_(a)PO₃H₂, —(CH₂)_(a)PO₃H⁻, —(CH₂)_(a)SO₃ ⁻, —(CH₂)_(a)SO₃H, —(CH₂)_(d)CO(CH₂CH₂O)_(c)R²³, —(CH₂)_(d)(CH₂CH₂O)_(c)R²⁴, —(CHCO₂H)_(a)CO₂H, —CH₂(CHNH₂)_(a)CH₂NR²⁵R²⁶, —CH₂(CHOH)_(a)CO₂H, —CH₂ (CHOH)_(a)R²⁷, —CH[(CH₂)_(b)NH₂]_(a)CH_(2—)OH, CH[(CH₂)_(b)NH₂]_(a)CO₂H, and —(CH₂)_(a)NR²⁸R²⁹; (AA) is a polypeptide chain comprising one or more natural or unnatural amino acids linked together by peptide bonds; (PS) is a sulfated or non-sulfated polysaccharide chain comprising one or more monosaccharide units connected by glycosidic linkages; and each ‘a’, and ‘d’ are independently chosen from 0 to 10, ‘c’ is chosen from 1 to 100 and each of ‘m’ and ‘n’ independently is an integer from 1 to 3; b. irradiating the subject's vasculature with non-ionizing radiation, wherein the radiation causes the compound to fluoresce; c. detecting the fluorescence of the compound in the subject's vasculature; and d. assessing the location of disease or injury in the subject's vasculature, based on the detected fluorescence.
 31. The method of claim 30, wherein the composition is administered intravenously.
 32. The method of claim 30, wherein the non-ionizing radiation has a wavelength of at least 350 nm.
 33. The method of claim 30, wherein the detected fluorescence of the compound in the subject's vasculature is measured over time.
 34. The method of claim 30, wherein the subject's pulmonary and cardiac vasculature is assessed.
 35. The method of claim 34, wherein assessing the subject's pulmonary and cardiac vasculature comprises identifying abnormalities chosen from stenosis, occlusions, aneurysms, and combinations thereof.
 36. The method of claim 35, wherein assessing the subject's pulmonary and cardiac vasculature comprises comparing the detected fluorescence in the subject's pulmonary and cardiac vasculature to that of normal pulmonary and cardiac vasculature under similar conditions.
 37. The method of claim 30, wherein the subject's eye vasculature is assessed.
 38. The method of claim 37, wherein assessing the subject's eye vasculature comprises identifying ocular abnormalities.
 39. The method of claim 38, wherein the ocular abnormalities are chosen from blood vessel architecture, ischemic spots, choroidal infarcts, Elschnig's spots, exudates, hemorrhages, and combinations thereof
 40. The method of claim 38, wherein the ocular abnormalities are chosen from vessel crossovers, vessel tortuosity, evidence of exudates, and combinations thereof.
 41. The method of claim 38, wherein assessing the subject's eye vasculature comprises comparing the detected fluorescence in the subject's eye to that of a normal eye under similar conditions.
 42. The method of claim 30, wherein said amino acids are chosen from α-amino acids, β-amino acids, and γ-amino acids. 